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Abstract: SA-PO1044

Role of WNK Aggregate Formation in Activating Na-Cl Cotransport

Session Information

  • Na+, K+, Cl-
    November 04, 2017 | Location: Hall H, Morial Convention Center
    Abstract Time: 10:00 AM - 10:00 AM

Category: Fluid, Electrolytes, and Acid-Base

  • 703 Na+, K+, Cl- Basic

Authors

  • Cuevas, Catherina A., Oregon Health & Science University, Portland, Oregon, United States
  • Miller, Lauren N, Oregon Health and Science University, Portland, Oregon, United States
  • Mutig, Kerim, Charite-Universitaetsmedizin Berlin, Berlin, Germany
  • Bachmann, Sebastian, Charité Universitätsmedizin Berlin, Berlin, Germany
  • Yang, Chao-Ling, Oregon Health and Science University, Portland, Oregon, United States
  • Ellison, David H., Oregon Health & Science University, Portland, Oregon, United States
Background

Activation of the thiazide-sensitive Na-Cl cotransporter (NCC) is essential to retain K+ in response to dietary K+ restriction and hypokalemia. Formation of WNK4/SPAK-OSR1 aggregates in distal convoluted tubule (DCT) cells is a common event that occurs when dietary K+ is low and NCC activity is high. While these aggegates bring together key signaling proteins required to activate NCC, it has not been clear whether WNK4 aggregate formation facilitates NCC activation or whether these represent stress features, or even autophagosomes.

Methods

Here, we fed C57/BL6 mice normal (NK, 0.8% K+) or potassium-deficient (LK, 0% K+) diets for 12 to 72 hours. Some mice were switched to high potssium diet (HK, 5%). Plasma electrolytes were determined with iSTAT. Protein aggregates containing WNK4, SPAK and ATG5 (autophagy marker) was assessed by light and electron microscopy.

Results

Plasma [K+] decreased with LK (3.3±0.3 vs 4.0±0.1 mmol/l); The abundance of phospho-NCC (T53) reached a maximum after only 12h of LK, with a striking increase in phospho SPAK-Ser373/OSR1-Ser325 at the apical membrane of DCT1 segments (identified with parvalbumin). Increased phospho NCC abundance at this time also correlated with appearance of phospho SPAK-Ser373/OSR1-Ser325 containing aggregates, but these aggregates did not contain WNK4, which appeared unaffected. WNK4 and ATG5, however, were clearly associated with aggregates in DCT seegments after 24h of LK diet and increased in a time-dependent fashion. By electron microscopy, the aggregates were not surrounded by membranes typical of autophagosomes. Potassium replenishment protocol (LK diet for 2 days followed by 2 additional days in HK) increased plasma [K+] (5.2± 0.1 mmol/l), as expected, and reduced phospho-NCC protein abundance. WNK4, SPAK and ATG5 aggregates were no longer detected in the DCT after this brief K+ replenishment. Basolateral K+ channels (Kir4.1) mediate [K+] sensing in DCT. In LK Kir4.1 KO mice, only occasional cells retaining Kir4.1 developed WNK4 aggregates.

Conclusion

WNK4-ATG5 aggregates are atypical, membraneless, dynamic structures that form after NCC is activated in response to LK, which require sensing through Kir4.1. WNK4 aggregate formation thus is not required for early NCC activation by LK, but these structures also are not typical autophagosomes.

Funding

  • NIDDK Support