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Abstract: FR-PO590

WNK Bodies Enable WNK4-Dependent Phosphorylation of SPAK/OSR1 for Their Apical Trafficking

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Thomson, Martin N., Charité Universitätsmedizin Berlin, Berlin, Germany
  • Yang, Chao-Ling, Oregon Health & Science University, Portland, Oregon, United States
  • Ellison, David H., Oregon Health & Science University, Portland, Oregon, United States
  • Mutig, Kerim, Charité Universitätsmedizin Berlin, Berlin, Germany
  • Bachmann, Sebastian, Charité Universitätsmedizin Berlin, Berlin, Germany
Background

The distal convoluted tubule (DCT) is vital for K+ homeostasis. Low plasma [K+] stimulates the apical Na+,Cl--cotransporter (NCC), limiting electrogenic K+ loss in the downstream tubule at the expense of increased NaCl retention and blood pressure. NCC is activated via phosphorylation by Ste-20-related proline/alanine-rich kinase (SPAK) and oxidative-stress-responsive kinase 1 (OSR1), two homologous substrates of with no lysine (WNK) kinases. During hypokalemic NCC activation, the human and rodent DCT develop cytoplasmic structures containing WNKs and SPAK/OSR1, termed WNK bodies. Their function is unclear. We hypothesized that WNK bodies serve as sites of SPAK/OSR1 activation, followed by trafficking of SPAK/OSR1 towards NCC.

Methods

To explore this hypothesis, we analyzed cellular distribution and phosphorylation of SPAK/OSR1 in different rodent models of hypokalemia (dietary K+ deprivation, genetic WNK4 deletion, and furosemide treatment) using high-resolution immunofluorescence and electron microscopy.

Results

Feeding mice (n=5) a K+-deficient diet for 10 days increased abundance of phosphorylated (p) SPAK/OSR1 at the apical DCT membrane and induced formation of WNK bodies enriched in pWNK and pSPAK/OSR1, suggesting that WNK bodies may facilitate phosphorylation steps. In contrast, only unphosphorylated WNK1 and SPAK/OSR1 were detected in WNK bodies of WNK4-deficient mice (n=3), which emphasizes the importance of WNK4 for SPAK/OSR1 activation in hypokalemia. Enlargement of WNK bodies along with reduction of apical SPAK/OSR1 in the WNK4-deficient DCT suggested that pSPAK/OSR1 might exit WNK bodies for apical trafficking towards NCC. To confirm this, we disrupted cellular trafficking machinery using the microtubule assembly inhibitor colchicine in rats receiving furosemide (n=4). Compared to rats receiving only furosemide (n=4), concomitant colchicine treatment resulted in accumulation of pSPAK/OSR1 in WNK bodies, along with reduced apical abundance of pSPAK/OSR1. Electron microscopy revealed that WNK bodies are membraneless, hypodense structures closely associated with microtubules.

Conclusion

In sum, our results indicate that WNK bodies are membraneless organelles performing SPAK/OSR1 activation for their subsequent apical trafficking, thereby linking plasma [K+] to NCC phosphorylation, NaCl balance and blood pressure.

Funding

  • NIDDK Support