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A five amino acids deletion in NKCC2 of C57BL/6 mice affects analysis of NKCC2 phosphorylation but does not impact kidney function


Moser, Sandra; Sugano, Yuya; Wengi, Agnieszka; Fisi, Viktoria; Lindtoft Rosenbaek, Lena; Mariniello, Marta; Loffing-Cueni, Dominique; McCormick, James A; Fenton, Robert A; Loffing, Johannes (2021). A five amino acids deletion in NKCC2 of C57BL/6 mice affects analysis of NKCC2 phosphorylation but does not impact kidney function. Acta Physiologica, 233:e13705.

Abstract

AIM

The phosphorylation level of the furosemide-sensitive Na$^{+}$ -K$^{+}$ -2Cl$^{-}$ cotransporter (NKCC2) in the thick ascending limb (TAL) is used as a surrogate marker for NKCC2 activation and TAL function. However, in mice, analyses of NKCC2 phosphorylation with antibodies against phosphorylated threonines 96 and 101 (anti-pT96/pT101) give inconsistent results. We aimed (a) to elucidate these inconsistencies and (b) to develop a phosphoform-specific antibody that ensures reliable detection of NKCC2 phosphorylation in mice.

METHODS

Genetic information, molecular biology, biochemical techniques and mouse phenotyping was used to study NKCC2 and kidney function in two commonly used mouse strains (ie 129Sv and in C57BL/6 mice). Moreover, a new phosphoform-specific mouse NKCC2 antibody was developed and characterized.

RESULTS

Amino acids sequence alignment revealed that C57BL/6 mice have a strain-specific five amino acids deletion (ΔF97-T101) in NKCC2 that diminishes the detection of NKCC2 phosphorylation with previously developed pT96/pT101 NKCC2 antibodies. Instead, the antibodies cross-react with the phosphorylated thiazide-sensitive NaCl cotransporter (NCC), which can obscure interpretation of results. Interestingly, the deletion in NKCC2 does not impact on kidney function and/or expression of renal ion transport proteins as indicated by the analysis of the F2 generation of crossbred 129Sv and C57BL/6 mice. A newly developed pT96 NKCC2 antibody detects pNKCC2 in both mouse strains and shows no cross-reactivity with phosphorylated NCC.

CONCLUSION

Our work reveals a hitherto unappreciated, but essential, strain difference in the amino acids sequence of mouse NKCC2 that needs to be considered when analysing NKCC2 phosphorylation in mice. The new pNKCC2 antibody circumvents this technical caveat.

Abstract

AIM

The phosphorylation level of the furosemide-sensitive Na$^{+}$ -K$^{+}$ -2Cl$^{-}$ cotransporter (NKCC2) in the thick ascending limb (TAL) is used as a surrogate marker for NKCC2 activation and TAL function. However, in mice, analyses of NKCC2 phosphorylation with antibodies against phosphorylated threonines 96 and 101 (anti-pT96/pT101) give inconsistent results. We aimed (a) to elucidate these inconsistencies and (b) to develop a phosphoform-specific antibody that ensures reliable detection of NKCC2 phosphorylation in mice.

METHODS

Genetic information, molecular biology, biochemical techniques and mouse phenotyping was used to study NKCC2 and kidney function in two commonly used mouse strains (ie 129Sv and in C57BL/6 mice). Moreover, a new phosphoform-specific mouse NKCC2 antibody was developed and characterized.

RESULTS

Amino acids sequence alignment revealed that C57BL/6 mice have a strain-specific five amino acids deletion (ΔF97-T101) in NKCC2 that diminishes the detection of NKCC2 phosphorylation with previously developed pT96/pT101 NKCC2 antibodies. Instead, the antibodies cross-react with the phosphorylated thiazide-sensitive NaCl cotransporter (NCC), which can obscure interpretation of results. Interestingly, the deletion in NKCC2 does not impact on kidney function and/or expression of renal ion transport proteins as indicated by the analysis of the F2 generation of crossbred 129Sv and C57BL/6 mice. A newly developed pT96 NKCC2 antibody detects pNKCC2 in both mouse strains and shows no cross-reactivity with phosphorylated NCC.

CONCLUSION

Our work reveals a hitherto unappreciated, but essential, strain difference in the amino acids sequence of mouse NKCC2 that needs to be considered when analysing NKCC2 phosphorylation in mice. The new pNKCC2 antibody circumvents this technical caveat.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Anatomy
04 Faculty of Medicine > Institute of Physiology
07 Faculty of Science > Institute of Physiology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > Physiology
Language:English
Date:September 2021
Deposited On:26 Aug 2021 08:48
Last Modified:01 Oct 2021 19:53
Publisher:Wiley-Blackwell Publishing, Inc.
ISSN:1748-1708
OA Status:Hybrid
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.1111/apha.13705
PubMed ID:34114742

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