Role of KLHL3 and dietary K⁺ in regulating KS-WNK1 expression

Abstract

The physiological role of the shorter isoform of WNK1 that is exclusively expressed in the kidney (KS-WNK1), with particular abundance in the distal convoluted tubule, remains elusive. KS-WNK1 despite lacking the kinase domain, is nevertheless capable of stimulating the NaCl cotransporter (NCC), apparently through activation of WNK4. It has recently been shown that a less severe form of the Familial Hyperkalemic Hypertension featuring only hyperkalemia is caused by missense mutations in the WNK1 acidic domain that preferentially affect CUL3-KLHL3 E3-induced degradation of KS-WNK1, rather than that of the full-length WNK1 (L-WNK1). Here we show that L-WNK1 is indeed less impacted by the CUL3-KLHL3 E3 ligase complex compared to KS-WNK1. We demonstrate that the unique 30 amino acid amino N-terminal fragment of KS-WNK1 is essential for its activating effect on NCC and recognition by KLHL3. We identify specific amino acid residues in this region critical for the functional effect of KS-WNK1 and KLHL3 sensitivity. To further explore this, we generated KLHL3-R528H knock-in mice that mimic human mutations causing Familial Hyperkalemic Hypertension. These mice revealed that the KLHL3 mutation specifically increased expression of KS-WNK1 in the kidney. We also observed that in wild type mice, expression of KS-WNK1 is only detectable after exposure to low potassium diet. These findings provide new insights into the regulation and function of KS-WNK1 by the CUL3-KLHL3 complex in DCT and indicate that this pathway is regulated by dietary K+ levels.

Molecular mechanisms for the modulation of blood pressure and potassium homeostasis by the distal convoluted tubule

“…The above‐mentioned role of KS‐WNK1 on NCC regulation in response to decreases in [K + ] e agrees with the recent observation that levels of KS‐WNK1 protein are greatly upregulated in mice on low K + diet (Ostrosky‐Frid et al , 2021). Interestingly, KS‐WNK1 protein levels were undetectable in blots of mice on normal diet, but were high in KLHL3‐R528H animals, suggesting that the low levels in wild‐type mice on normal chow may be due to a high rate of KLHL3‐targeted degradation, and that upregulation in the low K + diet may be due to decreased activity of the KLHL3‐CUL3 E3 ubiquitin ligase (Ishizawa et al , 2016).…”

“…These results agree with in vivo observations showing that in situations in which NCC is activated, like for instance, in mice fed with low K + diet or in KLHL3‐R528H mice, KS‐WNK1, WNK4, and SPAK form intracytoplasmic aggregates known as WNK bodies. Formation of these structures requires the presence of KS‐WNK1, since they are not observed in KS‐WNK1 knockout mice under similar conditions (Boyd‐Shiwarski et al , 2018; Thomson et al , 2020) (Ostrosky‐Frid et al , 2021).…”

WNK1 in the kidney

“…Their formation depends on the presence of KS-WNK1, as they are not observed in KS-WNK1 −/− mice on LKD [6]. These aggregates also contain WNK4, SPAK and OSR1, and they may serve as large complexes in which activation of the pathway is facilitated [5 ▪▪ ,6,23].…”

“…Finally, another interesting role of KS-WNK1 in the DCT is its participation in the formation of large cytoplasmic protein aggregates that have been named ‘WNK bodies’ [6]. These aggregates are formed in DCTs of mice in which the SPAK/OSR1-NCC pathway is overly active, like KLHL3 R528H/R528H mice [5 ▪▪ ] or wild-type mice on LKD [6,23,36]. Their formation depends on the presence of KS-WNK1, as they are not observed in KS-WNK1 −/− mice on LKD [6].…”

“…Translation of these transcripts initiates at a start codon encoded in exon 4a that is skipped in the long transcripts produced from the proximal promoter. Thus, the short transcripts that encode the short kidney-specific WNK1 (KS-WNK1) isoform contain a unique N-terminal segment of 30 amino acid residues that may confer certain functional properties [5 ▪▪ ,6]. Importantly, exons 1–4 of WNK1 encode part of the kinase domain, and thus, KS-WNK1 is catalytically inactive.…”