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Abstract: FR-OR23

Role of KLHL3-S433 Phosphorylation in Potassium Homeostasis in Mice

Session Information

Category: Fluid, Electrolytes, and Acid-Base Disorders

  • 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic


  • Ishizawa, Kenichi, Teikyo University School of Medicine, Itabashi-ku, Tokyo, Japan
  • Kaseda, Ken, Teikyo University School of Medicine, Itabashi-ku, Tokyo, Japan
  • Yamazaki, Osamu, Teikyo University School of Medicine, Itabashi-ku, Tokyo, Japan
  • Shibata, Shigeru, Teikyo University School of Medicine, Itabashi-ku, Tokyo, Japan

Kelch-like 3 (KLHL3) is a component of an E3 ubiquitin ligase complex that binds and degrades with-no-lysine kinases (WNKs) in the kidney. Previously, we reported that volume depletion and potassium(K) changes regulate KLHL3 function by altering phosphorylation at S433, thereby regulating fluid homeostasis. The current study was designed to demonstrate the physiological importance of S433 in KLHL3 function in vivo in mice.


S433 of KLHL3 was substituted to non-phosphorylatable Ala by CRISPR/Cas9 (KLHL3-S433A knock-in mice: KI). Baseline characteristics, as well as the response to K loading and to salt restriction, were compared between wild-type (WT) and KI. Efects of intestinal K binding by sodium zirconium cyclosilicate (SZC) was also tested.


KI showed a significant increase in plasma K levels that were attributable to the decreased renal excretion. KI also showed a decrease in plasma bicarbonate and an increase in blood pressure compared with WT. In KI, KLHL3 protein abundance (but not mRNA levels) was significantly reduced, which was accompanied by the increase in KS-WNK1, WNK4, and NCC, suggesting that S433 is important for the protein stability of KLHL3. In immunofluorescence, WNK1 puncta was observed not only in distal convoluted tubule but also in AQP2-positive collecting duct. In the renal cortex, K channel ROMK was decreased, not increased, in KI compared with WT. Given that KLHL3S433A can target WNK4 for degradation in vitro, we evaluated the response to K loading and to salt restriction. K challenge via 1% KCl did not alter plasma K in WT, while KI Plasma K increased to 1.7 mmol/L. In the kidney, NCC was significantly decreased by K in WT, whereas it remained unchanged in KI. Furthermore, although plasma K levels were significantly reduced by SZC in KI, NCC remained unaltered. Finally, increased aldosterone by a low-salt diet not alter plasma K in WT, whereas KI showed a significant reduction in plasma K in response to the same diet.


KLHL3-S433 regulates K homeostasis by controlling NCC and likely ROMK. The significant changes in plasma K levels in response to K loading and to volume depletion in KI indicate that the context-dependent regulation of renal K handling in high aldosterone states is compromised in this model, demonstrating the critical role of KLHL3S433 phosphorylation in the molecular basis of aldosterone paradox.