An emerging role for SPAK in NCC, NKCC, and blood pressure regulation.

The cloning of the related sodium chloride (NCC) and sodium potassium 2 chloride (NKCC) co-transporters in the early 1990s ushered in a new era for understanding the mechanisms of renal salt handling.1 Recent research has focused on the phosphoregulation of NCC and the two NKCC co-transporters: NKCC1, which is widely expressed, and NKCC2, which is renal specific. Two closely related kinases, STE20/SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1), seem to play a key role in NCC, NKCC1, and NKCC2 phosphorylation. All three co-transporters are phosphorylated by SPAK and OSR1 on N-terminal cytoplasmic serines and threonines that are conserved among NCC, NKCC1, and NKCC2, and phosphorylation of these residues results in co-transporter activation.2–5 The importance of these phosphorylation events for NCC activity was first demonstrated in Xenopus oocytes, in which mutation of these residues to nonphosphorylatable alanines reduced baseline NCC activity as well as stimulation of NCC by intracellular chloride depletion.6 Similar results were then observed in mammalian cells by Richardson et al.,7 who also showed that, like NCC, SPAK and OSR1 were activated by low intracellular chloride, and mutation of the SPAK/OSR1-binding site of NCC abolished NCC phosphorylation. These results suggest that specific stimuli activate SPAK and OSR1, which in turn activate NCC by phosphorylating N-terminal threonines and serines. The link between SPAK and NCC is strengthened by the report of Yang et al.8 in this issue of JASN. The authors generated SPAK null mice to demonstrate decreased levels of total NCC as well as decreased NCC phosphorylation on the Nterminal serines and threonines. This would predict a decrease in NCC activity. Consistent with this idea, SPAK null mice exhibit a phenotype similar to patients with Gitelman syndrome caused by mutations in NCC1: Low BP, elevated aldosterone levels, hypokalemia with increased fractional excretion of potassium, hypochloremia, hypomagnesemia with increased fractional excretion of magnesium, and hypocalciuria. Further evidence supporting a decrease in renal NCC activity is the lack of response to hydrochlorothiazide in the SPAK null mice.8 Because SPAK is also known to phosphorylate and activate NKCC1 and NKCC2, the authors investigated these co-transporters as well. NKCC1 phosphorylation in aorta is decreased in the SPAK null mice, and aortic rings from these animals demonstrate decreased contractile force in response to phenylephrine compared with aortic rings from wild-type mice.8 The authors propose that this deficiency contributes to the hypotension observed in the SPAK null mice, although more definitive proof of this would require tissue-specific SPAK knockout in the vasculature. Surprisingly, the authors also found that NKCC2 phosphorylation is increased. They attribute this finding to compensatory upregulation of OSR1. However, in two recent reports in which SPAK was unable to be activated9 or is missorted,10 phosphorylation of NKCC2 was decreased. Indeed, mice in which SPAK is missorted in thick ascending limb as a result of absence of the intracellular sorting receptor SORLA have a selective decrease in NKCC2 phosphorylation, whereas NCC phosphorylation is preserved.10 These mice have a phenotype reminiscent of human Bartter syndrome, which can be caused by loss-of-function mutations in NKCC2,1 suggesting that loss of NKCC2 phosphorylation results in decreased NKCC2 activity. What are the upstream events leading to NCC phosphorylation? Stimuli that activate renal salt retention, such as angiotensin II,11 aldosterone,4 vasopressin,12,13 and low-salt diet,4,14 result in N-terminal phosphorylation of NCC in rodents. Furthermore, these stimuli also result in the phosphorylation of SPAK/OSR1 on a conserved threonine in the activation loop.4,11,12,14 In vitro, cell culture, and Xenopus oocyte studies show that members of the with-no-lysine [K] (WNK) family of kinases, including WNK1 and WNK4 and likely WNK3, phosphorylate SPAK and OSR1 on the activation loop threonine, thereby activating SPAK and OSR1.2,3,15 This finding suggests that WNKs are positive regulators of SPAK and OSR1, which then are positive regulators of NCC and NKCCs. Consistent with the positive regulation of SPAK and OSR1 by WNKs, mice with a hypomorphic loss-of-function allele of WNK4 have modestly decreased levels of OSR1 phosphorylation (SPAK phosphorylation was not examined), decreased NCC Published online ahead of print. Publication date available at www.jasn.org.