Oi K et al. (2012), A minor role of WNK3 in regulating phosphorylat...

XB-ART-46295

Biol Open 2012 Feb 15;12:120-7. doi: 10.1242/bio.2011048.

Show Gene links Show Anatomy links

A minor role of WNK3 in regulating phosphorylation of renal NKCC2 and NCC co-transporters in vivo.

Oi K , Sohara E , Rai T , Misawa M , Chiga M , Alessi DR , Sasaki S , Uchida S .

???displayArticle.abstract???
Mutations in WNK1 and WNK4 kinase genes have been shown to cause a human hereditary hypertensive disease, pseudohypoaldosteronism type II (PHAII). We previously discovered that WNK kinases phosphorylate and activate OSR1/SPAK kinases that regulate renal SLC12A family transporters such as NKCC2 and NCC, and clarified that the constitutive activation of this cascade causes PHAII. WNK3, another member of the WNK kinase family, was reported to be a strong activator of NCC/NKCC2 when assayed in Xenopus oocytes, suggesting that WNK3 also plays a major role in regulating blood pressure and sodium reabsorption in the kidney. However, it remains to be determined whether WNK3 is in fact involved in the regulation of these transporters in vivo. To clarify this issue, we generated and analyzed WNK3 knockout mice. Surprisingly, phosphorylation and expression of OSR1, SPAK, NKCC2 and NCC did not decrease in knockout mouse kidney under normal and low-salt diets. Similarly, expression of epithelial Na channel and Na/H exchanger 3 were not affected in knockout mice. Na(+) and K(+) excretion in urine in WNK3 knockout mice was not affected under different salt diets. Blood pressure in WNK3 knockout mice was not lower under normal diet. However, lower blood pressure was observed in WNK3 knockout mice fed low-salt diet. WNK4 and WNK1 expression was slightly elevated in the knockout mice under low-salt diet, suggesting compensation for WNK3 knockout by these WNKs. Thus, WNK3 may have some role in the WNK-OSR1/SPAK-NCC/NKCC2 signal cascade in the kidney, but its contribution to total WNK kinase activity may be minimal.

???displayArticle.pubmedLink??? 23213404
???displayArticle.pmcLink??? PMC3507202
???displayArticle.link??? Biol Open
???displayArticle.grants??? [+]

Species referenced: Xenopus
Genes referenced: dct osr1 oxsr1 slc12a1 slc12a3 slc9a3 stk39 wnk1 wnk3 wnk4

???attribute.lit??? ???displayArticles.show???

	Fig. 1. Generation of WNK3 knockout mice.(A) Targeting strategy for Wnk3 gene interruption. The diagram shows the targeting construct, the wild-type WNK3 locus, and the targeted locus before and after Cre recombination. Three loxPs were inserted to flank exon 2 and the LacZ-Neo-selective marker. Exon 2 was deleted by mating the flox mice with CAG promoter Cre recombinase mice. (B) Verification of homologous recombination by PCR of genomic DNA derived from tails of mice. The primer set is indicated by dotted arrows in A. The 227-bp band and 339-bp band represent the wild-type allele and flox allele, respectively. (C) Genotyping PCR after Cre recombination, using a primer set indicated by solid arrows in A. A 516-bp PCR product was specific to the mutant allele. (D) Immunoblot of brain, kidney and testis homogenates probed with anti-WNK3 antibody. Absence of WNK3 protein in WNK3 knockout mouse was confirmed by immunoblotting in brain and testis. WNK3 was not detected by immunoblotting, even in wild-type mouse kidney, due to the low level of WNK3 protein expression in the kidney. (E) RT-PCR of brain, kidney and testis of wild-type and WNK3 knockout mice.
	Fig. 2. Segmental expression of WNK3 along mouse nephron.Primers for NHE3, NKCC2, NCC and ENaC were used as markers for each segment. WNK3 was positive in proximal tubules, thick ascending limb of Henle's loop, distal tubules and collecting ducts of wild type mouse. As a negative control, distal tubules and total kidney homogenate from WNK3 knockout mouse were used. PT, proximal tubule; TAL, thick ascending limb of Henle's loop; DCT, distal convoluted tubule; CD, collecting duct.
	Fig. 3. Urinary excretion of Na+ and K+ in WNK3 knockout mice.There were no significant differences in urine volume (A), urinary Na+ excretion (B) or K+ excretion (C) between WNK3 knockout mice (squares, n = 8) and their wild-type littermates (circles, n = 8). On day 0, mice were switched from low-salt diet (0.01% NaCl) to high-salt diet (4.0% NaCl). Before switching diet, mice were fed low-salt diet for 1 week. n.s. not significant.
	Fig. 4. Lower blood pressure in WNK3 knockout mice fed low-salt diet.(A) Blood pressure in WNK3 knockout mice under normal diet. Blood pressure was measured using a tail-cuff system. WNK3 knockout mice (n = 9) did not show significantly decreased systolic blood pressure, as compared to their wild-type littermates (n = 14). (B) Blood pressure in WNK3 knockout mice (n = 11) under low-salt diet. WNK3 knockout mice showed lower blood pressure, as compared to their wild-type littermates (n = 9). *P<0.05. n.s. not significant.
	Fig. 5. Expression and phosphorylation of NKCC2 and NCC in kidneys from WNK3 knockout mice under normal diet.(A) Representative immunoblots of total- and phosphorylated- NKCC2 and NCC in kidneys from wild-type and WNK3 knockout mice. (B) Densitometry analyses of expression and phosphorylation of NKCC2 and NCC in kidney from wild-type and WNK3 knockout mice. For densitometry analysis, values (n = 15) are expressed as ratios against the average of signals in the wild-type group. There were no significant decreases in the expression and phosphorylation of NKCC2 and NCC in kidneys from WNK3 knockout mice, as compared to wild-type littermates. (C) Representative immunoblots of p-OSR1 and p-SPAK in kidneys from wild-type and WNK3 knockout mice. (D) Densitometry analyses of p-OSR1 and p-SPAK in kidneys from wild-type and WNK3 knockout mice. For densitometry analysis, values (n = 15) are expressed as ratios against the average of signals in the wild-type group. There were no significant decreases in phosphorylation of OSR1 and SPAK in kidneys from WNK3 knockout mice, as compared to wild-type littermates. n.s. not significant.
	Fig. 6. Expression and phosphorylation of NKCC2 and NCC in kidneys from WNK3 knockout mice fed low-salt diet.(A) Representative immunoblots of total and phosphorylated NKCC2 and NCC in kidneys from wild-type and WNK3 knockout mice fed with low-salt diet. (B) Densitometry analyses of expression and phosphorylation of NKCC2 and NCC in kidneys from wild-type and WNK3 knockout mice fed low-salt diet. For densitometry analysis, values (n = 10) are expressed as ratios against the average of signals in the wild-type group. There were no significant decreases in the expression and phosphorylation of NKCC2 and NCC in kidneys from WNK3 knockout mice, as compared to wild-type littermates, even under low-salt diet. (C) Representative immunoblots of p-OSR1 and p-SPAK in kidneys from wild-type and WNK3 knockout mice fed low-salt diet. (D) Densitometry analyses of p-OSR1 and p-SPAK in kidneys from wild-type and WNK3 knockout mice fed low-salt diet. For densitometry analysis, values (n = 10) are expressed as ratios against the average of signals in the wild-type group. There were no significant decreases in phosphorylation of OSR1 and SPAK in kidneys from WNK3 knockout mice, as compared to wild-type littermates. n.s. not significant.
	Fig. 7. WNK1 and WNK4 were elevated in kidneys from WNK3 knockout mice fed low-salt diet.(A) Representative immunoblots of WNK1 and WNK4 in kidneys from wild-type and WNK3 knockout mice fed normal diet. (B) Densitometry analyses of WNK1 and WNK4 in kidneys from wild-type and WNK3 knockout mice fed normal diet. For densitometry analysis, values (n = 15) are expressed as ratios against the average of signals in the wild-type group. Expression of WNK4 was elevated in kidneys from WNK3 knockout mice, as compared to wild-type littermates. (C) Representative immunoblots of WNK1 and WNK4 in kidneys from wild-type and WNK3 knockout mice fed low-salt diet. (D) Densitometry analyses of WNK1 and WNK4 in kidneys from wild-type and WNK3 knockout mice fed low-salt diet. For densitometry analysis, values (n = 10) are expressed as ratios against the average of signals in the wild-type group. Expression of WNK1 and WNK4 was elevated in kidneys from WNK3 knockout mice, as compared to wild-type littermates. *P<0.05. n.s. not significant.

References [+] :

Achard, Phenotypic and genetic heterogeneity of familial hyperkalaemic hypertension (Gordon syndrome). 2001, Pubmed

Achard, Phenotypic and genetic heterogeneity of familial hyperkalaemic hypertension (Gordon syndrome). 2001, Pubmed
Akar, Contractile regulation of the Na(+)-K(+)-2Cl(-) cotransporter in vascular smooth muscle. 2001, Pubmed
Anselmo, WNK1 and OSR1 regulate the Na+, K+, 2Cl- cotransporter in HeLa cells. 2006, Pubmed
Chiga, Dietary salt regulates the phosphorylation of OSR1/SPAK kinases and the sodium chloride cotransporter through aldosterone. 2008, Pubmed
Chiga, Phenotypes of pseudohypoaldosteronism type II caused by the WNK4 D561A missense mutation are dependent on the WNK-OSR1/SPAK kinase cascade. 2011, Pubmed
Cruz-Rangel, Similar effects of all WNK3 variants on SLC12 cotransporters. 2011, Pubmed , Xenbase
Gamba, Molecular physiology and pathophysiology of electroneutral cation-chloride cotransporters. 2005, Pubmed
Garg, Effect of the Na-K-2Cl cotransporter NKCC1 on systemic blood pressure and smooth muscle tone. 2007, Pubmed
Glover, Renal and brain isoforms of WNK3 have opposite effects on NCCT expression. 2009, Pubmed , Xenbase
Gordon, Syndrome of hypertension and hyperkalemia with normal glomerular filtration rate. 1986, Pubmed
Haas, The Na-K-Cl cotransporters. 1998, Pubmed
Holden, Cloning, genomic organization, alternative splicing and expression analysis of the human gene WNK3 (PRKWNK3). 2004, Pubmed
Kahle, WNK3 modulates transport of Cl- in and out of cells: implications for control of cell volume and neuronal excitability. 2005, Pubmed , Xenbase
Kim, Salt sensitivity of blood pressure in NKCC1-deficient mice. 2008, Pubmed
Meyer, Decreased blood pressure and vascular smooth muscle tone in mice lacking basolateral Na(+)-K(+)-2Cl(-) cotransporter. 2002, Pubmed
Moriguchi, WNK1 regulates phosphorylation of cation-chloride-coupled cotransporters via the STE20-related kinases, SPAK and OSR1. 2005, Pubmed
Naito, Regulation of WNK1 kinase by extracellular potassium. 2011, Pubmed
Nomura, Generation and analyses of R8L barttin knockin mouse. 2011, Pubmed
Ohno, Immunolocalization of WNK4 in mouse kidney. 2011, Pubmed
Ohta, Targeted disruption of the Wnk4 gene decreases phosphorylation of Na-Cl cotransporter, increases Na excretion and lowers blood pressure. 2009, Pubmed
Pacheco-Alvarez, The Na+:Cl- cotransporter is activated and phosphorylated at the amino-terminal domain upon intracellular chloride depletion. 2006, Pubmed , Xenbase
Pedersen, Vasopressin induces phosphorylation of the thiazide-sensitive sodium chloride cotransporter in the distal convoluted tubule. 2010, Pubmed
Ponce-Coria, Regulation of NKCC2 by a chloride-sensing mechanism involving the WNK3 and SPAK kinases. 2008, Pubmed , Xenbase
Rafiqi, Role of the WNK-activated SPAK kinase in regulating blood pressure. 2010, Pubmed
Richardson, Activation of the thiazide-sensitive Na+-Cl- cotransporter by the WNK-regulated kinases SPAK and OSR1. 2008, Pubmed
Richardson, The regulation of salt transport and blood pressure by the WNK-SPAK/OSR1 signalling pathway. 2008, Pubmed
Richardson, Regulation of the NKCC2 ion cotransporter by SPAK-OSR1-dependent and -independent pathways. 2011, Pubmed
Rinehart, WNK3 kinase is a positive regulator of NKCC2 and NCC, renal cation-Cl- cotransporters required for normal blood pressure homeostasis. 2005, Pubmed , Xenbase
Sakai, A transgenic mouse line that retains Cre recombinase activity in mature oocytes irrespective of the cre transgene transmission. 1997, Pubmed
San-Cristobal, WNK3 and WNK4 amino-terminal domain defines their effect on the renal Na+-Cl- cotransporter. 2008, Pubmed , Xenbase
San-Cristobal, Angiotensin II signaling increases activity of the renal Na-Cl cotransporter through a WNK4-SPAK-dependent pathway. 2009, Pubmed , Xenbase
Schambelan, Mineralocorticoid-resistant renal hyperkalemia without salt wasting (type II pseudohypoaldosteronism): role of increased renal chloride reabsorption. 1981, Pubmed
Sohara, Acute insulin stimulation induces phosphorylation of the Na-Cl cotransporter in cultured distal mpkDCT cells and mouse kidney. 2011, Pubmed
Sohara, Pathogenesis and treatment of autosomal-dominant nephrogenic diabetes insipidus caused by an aquaporin 2 mutation. 2006, Pubmed
Talati, Effect of angiotensin II on the WNK-OSR1/SPAK-NCC phosphorylation cascade in cultured mpkDCT cells and in vivo mouse kidney. 2010, Pubmed
Vallon, Expression and phosphorylation of the Na+-Cl- cotransporter NCC in vivo is regulated by dietary salt, potassium, and SGK1. 2009, Pubmed
Veríssimo, Protein kinase WNK3 increases cell survival in a caspase-3-dependent pathway. 2006, Pubmed
Vitari, The WNK1 and WNK4 protein kinases that are mutated in Gordon's hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases. 2005, Pubmed
Vitari, Functional interactions of the SPAK/OSR1 kinases with their upstream activator WNK1 and downstream substrate NKCC1. 2006, Pubmed
Wilson, Human hypertension caused by mutations in WNK kinases. 2001, Pubmed
Yang, Molecular pathogenesis of pseudohypoaldosteronism type II: generation and analysis of a Wnk4(D561A/+) knockin mouse model. 2007, Pubmed
Yang, The thiazide-sensitive Na-Cl cotransporter is regulated by a WNK kinase signaling complex. 2007, Pubmed , Xenbase
Yang, SPAK-knockout mice manifest Gitelman syndrome and impaired vasoconstriction. 2010, Pubmed
Zambrowicz, Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention. 2003, Pubmed
de Los Heros, WNK3 bypasses the tonicity requirement for K-Cl cotransporter activation via a phosphatase-dependent pathway. 2006, Pubmed , Xenbase
van der Lubbe, Angiotensin II induces phosphorylation of the thiazide-sensitive sodium chloride cotransporter independent of aldosterone. 2011, Pubmed