Vascular Phenotype of STE20/SPS1-related Proline/Alanine-rich Kinase (SPAK) Targeted Mouse Model of Gitelman Syndrome

K Siew, JJ Maguire, AP Davenport, KM O’Shaughnessy. Clinical Pharmacology Unit, Department of Medicine, University of Cambridge, Cambridge, UK

Gitelman syndrome is a rare monogenetic disease with a salt-wasting hypotensive phenotype caused by reduced renal thiazide-sensitive Na+-Cl- cotransporter (NCC) activity. SPAK and oxidative stress-response kinase-1 (OSR1) colocalise with NCC and the bumetanide-sensitive Na+-K+-2Cl- cotransporter (NKCC1) and increase their activity by phosphorylation. We have previously generated a SPAK loss-of-function knock-in (SPAK-KI) mouse that recapitulates human Gitelman syndrome by reducing phospho-NCC (1). SPAK knock-out (SPAK-KO) mice have also been generated independently, though with subtle phenotypic differences (2,3). In particular, SPAK-KO mice exhibit 50% reduction in aortic contractility compared to wildtypes (WT), due to a 70% decrease in phospho-NKCC1; the activity of which normally increases contractility (2). We hypothesised that SPAK-KI mice would also have reduced contractility compared to WT littermates. The aim of this study was to investigate the vascular phenotype of protein-intact SPAK-KI mice in comparison to the published phenotype of protein-ablated SPAK-KO mice.

Age-matched adult C57Bl/6 WT and SPAK-KI mice (both n=11) were euthanised by cervical dislocation. Rings of endothelium-intact thoracic aorta (1.5-2mm) were mounted in a wire myograph for isometric tension recordings as described previously (4). To assess the influence of altered NKCC1 activity on contractility, aortae were incubated with either 10μM bumetanide (Bumet) or 0.1%v/v ethanol (EtOH) vehicle control. Cumulative concentration-response curves were generated to the phenylephrine (10-9M-10-4M) and data analysed using a 4-parameter logistic function (GraphPad Prism 5) to determine values of pD2 (-log10 of the molar concentration producing half maximal response) and maximum response (EMAX, mN/mm). n-Values are the number of mice from which tissues were obtained.

In contrast to the reported attenuated response to phenylephrine in SPAK-KO mice we found no significant difference (P>0.05) in responsiveness between SPAK-KI and WT littermates in the absence or presence of bumetanide (Table 1).

Table 1

Data are mean ± SEM. One-way repeated ANOVA with Bonferroni’s post hoc test, significance at P<0.05

In conclusion, vascular contractility remains intact in the SPAK-KI mice but is reportedly reduced in the SPAK-KO mice that we suggest results from differences in model design and generation. Recent data support a role for SPAK-dependent stabilisation of OSR1. Therefore the intact mutant protein of SPAK-KI mice may continue to stabilise OSR1, and thus maintain vascular phospho-NKCC1 levels by compensating for loss of SPAK phosphorylation activity. Additionally, newly discovered SPAK isoforms, absent in SPAK-KO but present in SPAK-KI (3), may play yet unexplored roles in vascular contractility.

1 Rafiqi FH et al, EMBO Mol Med 2:63, 2010

2 Yang SS et al, J Am Soc Nephrol 21:1868, 2010

3 Grimm PR et al, J Biol Chem 287:37868, 2012

4 Maguire JJ et al, Exp Biol Med (Maywood) 231:806, 2006