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Abstract: FR-OR062

SPAK Is Rapidly Dephosphorylated in the Distal Convoluted Tubule during Acute Potassium Loading

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Mukherjee, Anindit, Oregon Health Science University, Portland, Oregon, United States
  • Yang, Chao-Ling, Oregon Health and Science University , Portland, Oregon, United States
  • Ellison, David H., Oregon Health & Science University, Portland, Oregon, United States
Background

The thiazide-sensitive sodium chloride cotransporter (NCC) of the distal convoluted tubule (DCT) is phosphorylated and activated by WNK acting through SPAK. High extracellular potassium leads to rapid NCC dephosphorylation and inactivation. However, the molecular mechanism remains unknown. Here, we examined the roles of SPAK in this process: specifically, testing the hypothesis that SPAK is dephosphorylated by high extracellular K in cultured cells and in vivo.

Methods

1) SPAK-transfected HEK293 cells were treated with 0mM to 10Mm (high) KCl, for one hour. Cell extracts were analyzed by western blot. 2) Kidney slices were incubated for 30 minutes in 0 Mm K to increase NCC phosphorylation and then treated with 3mM or 10mM K for 30 minutes. Slices were snap frozen. 3) C57/BL6 mice were gavaged with either vehicle or 3% KCl for 15 mins. Blood collected was used for electrolyte measurement by iSTAT. One kidney was processed for western blot and the other kidney was either perfused for IF or cortex-rich kidney poles were isolated and snap frozen.

Results

In HEK293 cells, raising medium [K+] for one hour led to a reduction in transfected and endogenous SPAK phosphorylation at S383. This in vitro finding was replicated ex vivo in kidney slices by western blot analysis. We saw parallel decrease in NCC and SPAK phosphorylation levels in kidney slices incubated in high K (10mM) vs normal K (3mM) buffer. We used a model similar to Loffing and colleagues to test whether SPAK dephosphorylation by high K+ occurs in a physiologically relevant setting. C57/BL6 mice gavaged with 3% KCl for 15 minutes showed an increase in plasma [K+] and a corresponding decrease in SPAK phosphorylation in the cortex, but not in whole kidney. Immunofluorescence studies also showed decreased SPAK-S383 primarily in parvalbumin-positive ‘early’ DCT1 upon K loading. Cytosolic total-SPAK puncta observed in control kidneys rapidly disappeared after K loading, suggesting a rapid alteration in SPAK localization as well as phosphorylation by extracellular K.

Conclusion

Although prior work suggested that SPAK is not involved in rapid NCC dephosphorylation, the current data indicate that SPAK is rapidly dephosphorylated in vivo primarily along the DCT during short term K treatment. Further work will be necessary to establish that this effect is essential for rapid NCC dephosphorylation.

Funding

  • NIDDK Support