Title

SPAK (Stk39) is Involved in NKCC2 Phosphorylation and Salt-sensitive Hypertension in Dahl Salt-sensitive Rats

Document Type

Conference Proceeding

Publication Date

2018

Publication Title

FASEB Journal

Abstract

In the thick ascending limb (TAL), NaCl reabsorption is mediated by the Na/K/2Cl cotransporter NKCC2, which plays a crucial role in the regulation of blood pressure. NKCC2 activity is partly controlled by phosphorylation via STE20/SPS1-related proline-alanine-rich kinase (SPAK, gene name Stk39) at Thr96 and Thr101 residues in the NH2-terminus of NKCC2. Previously, we found that NKCC2 phosphorylation was 5-fold higher in TALs from Dahl salt-sensitive (DSS) rats, suggesting that this pathway may be related to the salt-sensitivity in this model. However, the kinase responsible for NKCC2 hyperphosphorylation is unknown. In addition, the role of SPAK in the development of salt-sensitive hypertension remains unclear. We hypothesized that SPAK is involved in salt-sensitive hypertension in part by increasing NKCC2 phosphorylation during normal or high salt diet. To test this, we generated SPAK knockout (KO) rats in a DSS genetic background via Crispr/Cas9 targeting of exon 5 in collaboration with the Gene Editing Rat Resource Center (Medical College of Wisconsin). Gene deletion was confirmed by sequencing and RT-PCR. We used radiotelemetry to continuously measure mean blood pressure (MBP) in these rats on normal Na (0.22% Na chow) or high salt diet (4% Na chow). At baseline, SPAK KO rats had lower MBP than wild-type DSS (WT) rats (SPAK KO: 117±1 mmHg; WT: 127±2 mmHg; p<0.05, n=7 each). After 4 weeks on high salt diet, MBP rose in all rats reaching 137±3 in SPAK KO and 154±3 mmHg in WT rats (p<0.05). The increase in MBP after switching rats from normal to high salt was lower in SPAK KO than WT rats; at 7 days (8±1 vs 13±1 mmHg, p<0.05), 14 days (12±2 vs 17±2 mmHg, p<0.05), and 28 days (20±3 vs 27±2 mmHg, p<0.05). We then explored total and phosphorylated-NKCC2 (Thr96 and Thr101) expression in isolated TALs by Western blot. In rats fed normal salt (0.22% Na), phosphorylated-NKCC2 (corrected over total NKCC2 expression) was lower in TALs isolated from SPAK KO (WT: 100; SPAK KO: 39±25%; p<0.05, n=4) whereas total NKCC2 expression (corrected by GAPDH) was not different between both groups (WT: 100; SPAK KO: 103±17%; n=5). After 4 weeks on high-salt (4% Na), phosphorylated-NKCC2 was also lower in TALs isolated from SPAK KO (WT: 100; SPAK KO: 38±13%; p<0.05, n=4) whereas total NKCC2 expression (corrected by GAPDH) was not different between both groups (WT: 100; SPAK KO: 113±22%; n=4). We conclude that SPAK KO rats exhibit a lower blood pressure at baseline and decreased salt sensitivity. This may be in part caused by lower NKCC2 phosphorylation in TALs of SPAK KO DSS rats. Our findings support an important role for SPAK in NKCC2 hyperphosphorylation and salt sensitive hypertension in DSS rats.

Volume

32

Issue

1 Suppl

First Page

620.9

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