Potassium depletion stimulates Na-Cl cotransporter via phosphorylation and inactivation of the ubiquitin ligase Kelch-like 3

Abstract

Kelch-like 3 (KLHL3) is a component of an E3 ubiquitin ligase complex that regulates blood pressure by targeting With-No-Lysine (WNK) kinases for degradation. Mutations in KLHL3 cause constitutively increased renal salt reabsorption and impaired K+ secretion, resulting in hypertension and hyperkalemia. Although clinical studies have shown that dietary K+ intake affects blood pressure, the mechanisms have been obscure. In this study, we demonstrate that the KLHL3 ubiquitin ligase complex is involved in the low-K+-mediated activation of Na-Cl cotransporter (NCC) in the kidney. In the distal convoluted tubules of mice eating a low-K+ diet, we found increased KLHL3 phosphorylation at S433 (KLHL3S433-P), a modification that impairs WNK binding, and also reduced total KLHL3 levels. These changes are accompanied by the accumulation of the target substrate WNK4, and activation of the downstream kinases SPAK (STE20/SPS1-related proline-alanine-rich protein kinase) and OSR1 (oxidative stress-responsive 1), resulting in NCC phosphorylation and its accumulation at the plasma membrane. Increased phosphorylation of S433 was explained by increased levels of active, phosphorylated protein kinase C (but not protein kinase A), which directly phosphorylates S433. Moreover, in HEK cells expressing KLHL3 and WNK4, we showed that the activation of protein kinase C by phorbol 12-myristate 13-acetate induces KLHL3S433-P and increases WNK4 levels by abrogating its ubiquitination. These data demonstrate the role of KLHL3 in low-K+-mediated induction of NCC; this physiologic adaptation reduces distal electrogenic Na+ reabsorption, preventing further renal K+ loss but promoting increased blood pressure.

C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity

“…6, A and B). The latter result is consistent with a previous report (34), and the changes observed are probably due to a decrease in KLHL3-CUL3-RING-mediated WNK4 degradation. However, no changes in the relative abundance…”

30 YEARS OF THE MINERALOCORTICOID RECEPTOR: Mineralocorticoid receptor and NaCl transport mechanisms in the renal distal nephron

“…To evaluate the possible involvement of KLHL3/ CUL3-based ubiquitin ligase in low-K + -mediated NCC induction, we recently analyzed KLHL3 levels in mice on a low-K + diet (Ishizawa et al 2016). Notably, we found that KLHL3 S433-P levels were increased whereas total KLHL3 levels were decreased in the kidney by K + depletion.…”

“…Thus, the increase in WNK levels by KLHL3 inactivation and WNK kinase activation induced by the change in intracellular Cl − levels synergistically regulate NCC (Fig. 4) (Terker et al 2015b, Ishizawa et al 2016. A recent study also reported that a Cl − -and SPAK/OSR1-independent pathway activates NCC in hypokalemia (Penton et al 2016).…”

“…This was the first demonstration of the involvement of KLHL3/CUL3 ubiquitin ligase in aldosteroneindependent, low-K + -mediated NCC induction (Fig. 4) (Ishizawa et al 2016).…”

Renal potassium physiology: integration of the renal response to dietary potassium depletion

“…109,110 K þ depletion has also been shown to activate protein kinase C, resulting in increased phosphorylation of KLHL3 at Ser433. 111 The T-shaped, dashed modified arrow structures represent removal of an inhibitory effect. Hypokalemia is associated with an increase in Ang II, which converts WNK4 to an NCC-activating kinase.…”