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.

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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 .

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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.

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

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	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, nâ=â8) and their wild-type littermates (circles, nâ=â8). 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 (nâ=â9) did not show significantly decreased systolic blood pressure, as compared to their wild-type littermates (nâ=â14). (B) Blood pressure in WNK3 knockout mice (nâ=â11) under low-salt diet. WNK3 knockout mice showed lower blood pressure, as compared to their wild-type littermates (nâ=â9). *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 (nâ=â15) 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 (nâ=â15) 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 (nâ=â10) 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 (nâ=â10) 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 (nâ=â15) 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 (nâ=â10) 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.

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