Abstract
Mutations within the with-no-K(Lys) (WNK) kinases cause Gordon's syndrome characterized by hypertension and hyperkalaemia. WNK kinases phosphorylate and activate the STE20/SPS1-related proline/alanine-rich kinase (SPAK) protein kinase, which phosphorylates and stimulates the key Na+:Cl− cotransporter (NCC) and Na+:K+:2Cl− cotransporters (NKCC2) cotransporters that control salt reabsorption in the kidney. To define the importance of this pathway in regulating blood pressure, we generated knock-in mice in which SPAK cannot be activated by WNKs. The SPAK knock-in animals are viable, but display significantly reduced blood pressure that was salt-dependent. These animals also have markedly reduced phosphorylation of NCC and NKCC2 cotransporters at the residues phosphorylated by SPAK. This was also accompanied by a reduction in the expression of NCC and NKCC2 protein without changes in messenger RNA (mRNA) levels. On a normal Na+-diet, the SPAK knock-in mice were normokalaemic, but developed mild hypokalaemia when the renin–angiotensin system was activated by a low Na+-diet. These observations establish that SPAK plays an important role in controlling blood pressure in mammals. Our results imply that SPAK inhibitors would be effective at reducing blood pressure by lowering phosphorylation as well as expression of NCC and NKCC2. See accompanying Closeup by Maria Castañeda-Bueno and Gerald Gamba (DOI 10.1002/emmm.200900059).
Highlights
We demonstrate that preventing SPS1-related proline/alanine-rich kinase (SPAK) activation by WNK kinases significantly reduced blood pressure by suppressing expression and phosphorylation of the Naþ:ClÀ cotransporter (NCC) and NKCC2 ion cotransporters
Generation of knock-in mice Knock-in mice in which the T-loop Thr residue in SPAK (Thr243) and oxidative stress-responsive kinase 1 (OSR1) (Thr185) were mutated to Ala to prevent activation by WNK isoforms were generated as described in Supporting Information Fig 1
Reduced phosphorylation and expression of NKCC1 in kidney, but not in other tissues of SPAK knock-in mice We observed a $30% reduction in the expression of NKCC1 in kidney extracts derived from SPAK243A/243A mice compared to heterozygous or wild type mice (Fig 4C)
Summary
(4) Division of Medical Sciences, Centre for Cardiovascular and Lung Biology, Ninewells Hospital and Medical School, University of Dundee, Dundee, Scotland, UK. The bestcharacterized WNK substrates comprise the STE20/SPS1related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1), which are activated following their phosphorylation by WNK1 or WNK4 (Delpire & Gagnon, 2008; Richardson & Alessi, 2008; Vitari et al, 2005). These WNK isoforms interact directly with SPAK as well as OSR1 and phosphorylate these enzymes at two conserved www.embomolmed.org
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