SPAK-Knockout Mice Manifest Gitelman Syndrome and Impaired Vasoconstriction

Abstract

Polymorphisms in the gene encoding sterile 20/SPS1-related proline/alanine-rich kinase (SPAK) associate with hypertension susceptibility in humans. SPAK interacts with WNK kinases to regulate the Na ϩ -K ϩ -2Cl Ϫ and NaMutations in WNK1/4 and N(K)CC can cause changes in BP and dyskalemia in humans, but the physiologic role of SPAK in vivo is unknown. We generated and analyzed SPAK-null mice by targeting disruption of exons 9 and 10 of SPAK. Compared with SPAK ϩ/ϩ littermates, SPAK ϩ/Ϫ mice exhibited hypotension without significant electrolyte abnormalities, and SPAK Ϫ/Ϫ mice not only exhibited hypotension but also recapitulated Gitelman syndrome with hypokalemia, hypomagnesemia, and hypocalciuria. In the kidney tissues of SPAK Ϫ/Ϫ mice, the expression of total and phosphorylated (p-)NCC was markedly decreased, but that of p-OSR1, total NKCC2, and p-NKCC2 was significantly increased. We observed a blunted response to thiazide but normal response to furosemide in SPAK Ϫ/Ϫ mice. In aortic tissues, total NKCC1 expression was increased but p-NKCC1 was decreased in SPAK-deficient mice. Both SPAK ϩ/Ϫ and SPAK Ϫ/Ϫ mice had impaired responses to the selective ␣ 1 -adrenergic agonist phenylephrine and the NKCC1 inhibitor bumetanide, suggesting that impaired aortic contractility may contribute to the hypotension of SPAKnull mice. In summary, SPAK-null mice have defects of NCC in the kidneys and NKCC1 in the blood vessels, leading to hypotension through renal salt wasting and vasodilation. SPAK may be a promising target for antihypertensive therapy. 21: 186821: -187721: , 201021: . doi: 10.1681 Sterile 20/SPS1-related proline/alanine-rich kinase (SPAK) 1,2 and oxidative stress-responsive kinase 1 (OSR1) 3 are serine/threonine kinases that share high homology in both their N-terminal catalytic and Cterminal regulatory domains and are widely distributed in the brain, pancreas, heart and kidney. Gene mutations of the NCC in the distal convoluted tubules (DCTs) and NKCC2 in the thick ascending limb of the loop of Henle (TAL) cause autosomal recessive Gitelman syndrome (GS) 12 and Bartter syndrome (BS), 13 respectively. These congenital renal tubular disorders are characterized by J Am Soc Nephrol

Short Forms of Ste20-related Proline/Alanine-rich Kinase (SPAK) in the Kidney Are Created by Aspartyl Aminopeptidase (Dnpep)-mediated Proteolytic Cleavage

“…Although in sensory neurons the two kinases seem to fulfill a redundant function (12) based on the phenotypes of SPAK and kidney-specific OSR1 knock-out mice, the two kinases in kidney seem to have very distinct roles. Indeed, although the SPAK knock-out mouse reveals a primary role for SPAK in regulating the Na-Cl cotransporter in the distal convoluted tubule (13)(14)(15)(16), the kidney-specific OSR1 knock-out mouse displays a phenotype that is consistent with disruption of NKCC2 function in the TAL (17). An intriguing observation from the SPAK knock-out mouse is that NKCC2 in the TAL is hyperphosphorylated (13,14,16), which would be inconsistent if the kinase was phosphorylating this cotransporter under normal conditions.…”

“…Indeed, although the SPAK knock-out mouse reveals a primary role for SPAK in regulating the Na-Cl cotransporter in the distal convoluted tubule (13)(14)(15)(16), the kidney-specific OSR1 knock-out mouse displays a phenotype that is consistent with disruption of NKCC2 function in the TAL (17). An intriguing observation from the SPAK knock-out mouse is that NKCC2 in the TAL is hyperphosphorylated (13,14,16), which would be inconsistent if the kinase was phosphorylating this cotransporter under normal conditions. In the kidney medulla several smaller SPAK fragments are observed by Western blot analysis, and these fragments have been postulated to act as negative regulators of OSR1 function in the thick ascending limb of Henle (14,16,18).…”

Generation of Hypertension-Associated STK39 Polymorphism Knockin Cell Lines With the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 System

“…We and other groups have previously shown that the with-nolysine kinase-SPAK-Na-Cl cotransporter/NKCC signaling cascade plays a pivotal role in BP regulation in the kidney and aorta. [17][18][19][20] This study suggests that subjects with rs3754777 develop an activation of this cascade primarily based on increased SPAK transcription, leading to hypertension. A previous study showed a link between genetic variants and saltsensitive BP elevation in Korean population 32 and identified this SNP as one of the variants associated with salt sensitivity.…”

“…[17][18][19][20][21] In particular, phosphorylation and activation of the SLC12A cotransporters (Na-Cl cotransporter/Na-K-Cl cotransporter 1/2 [NKCC1/NKCC2]) have been shown to depend completely on SPAK and oxidative stress responsive 1 activity. [18][19][20][21] Thus, STK39 polymorphisms have been speculated to modulate the function of these cotransporters, resulting in BP elevation. 4 However, the biochemical functions of these polymorphisms and the mechanisms of their contribution to hypertension remain to be clarified.…”

“…41824, respectively). Using the CRISPR Design Tool (http://crisp.mit.edu), 23-bp sequences of 2 pairs of single-guide RNAs (sgRNAs A and B) for the double nicking-targeted sites flanking SNP were designed, according to the (N) 20 NGG rule. The 2 pairs of 60-nt single-stranded DNA oligonucleotides (ssODNs) based on sgRNAs A and B (Table 1) were annealed, and the 2 annealed 100-bp double-stranded DNAs were incorporated into gRNA cloning vectors using Gibson Assembly (New England Biolabs Inc) according to the sgRNA synthesis protocol provided by the Church laboratory at Harvard Medical School.…”