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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

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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 et al.

I: Acta Physiologica (Print), Bind 233, Nr. 1, e13705, 09.2021.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

Harvard

Moser, S, Sugano, Y, Wengi, A, Fisi, V, Lindtoft Rosenbaek, L, Mariniello, M, Loffing-Cueni, D, McCormick, JA, Fenton, RA & Loffing, J 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 (Print), bind 233, nr. 1, e13705. https://doi.org/10.1111/apha.13705

APA

Moser, S., Sugano, Y., Wengi, A., Fisi, V., Lindtoft Rosenbaek, L., Mariniello, M., Loffing-Cueni, D., McCormick, J. A., Fenton, R. A., & Loffing, J. (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 (Print), 233(1), [e13705]. https://doi.org/10.1111/apha.13705

CBE

Moser S, Sugano Y, Wengi A, Fisi V, Lindtoft Rosenbaek L, Mariniello M, Loffing-Cueni D, McCormick JA, Fenton RA, Loffing J. 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 (Print). 233(1):Article e13705. https://doi.org/10.1111/apha.13705

MLA

Vancouver

Author

Moser, Sandra ; Sugano, Yuya ; Wengi, Agnieszka et al. / A five amino acids deletion in NKCC2 of C57BL/6 mice affects analysis of NKCC2 phosphorylation but does not impact kidney function. I: Acta Physiologica (Print). 2021 ; Bind 233, Nr. 1.

Bibtex

@article{3db88d288d8c4c74967bc92ddbc167bb,
title = "A five amino acids deletion in NKCC2 of C57BL/6 mice affects analysis of NKCC2 phosphorylation but does not impact kidney function",
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. ",
keywords = "NKCC2, ion homeostasis, kidney, phosphorylation, strain differences, K-CL COTRANSPORTER, SPAK, PROTEIN, SALT, DISTAL CONVOLUTED TUBULE, NACL COTRANSPORTER, STRAIN DIFFERENCES, DEOXYCORTICOSTERONE ACETATE, SODIUM-CHLORIDE COTRANSPORTER, THICK ASCENDING LIMB",
author = "Sandra Moser and Yuya Sugano and Agnieszka Wengi and Viktoria Fisi and {Lindtoft Rosenbaek}, Lena and Marta Mariniello and Dominique Loffing-Cueni and McCormick, {James A} and Fenton, {Robert A} and Johannes Loffing",
note = "{\textcopyright} 2021 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.",
year = "2021",
month = sep,
doi = "10.1111/apha.13705",
language = "English",
volume = "233",
journal = "Acta Physiologica (Print)",
issn = "1748-1708",
publisher = "Wiley-Blackwell Publishing Ltd.",
number = "1",

}

RIS

TY - JOUR

T1 - A five amino acids deletion in NKCC2 of C57BL/6 mice affects analysis of NKCC2 phosphorylation but does not impact kidney function

AU - Moser, Sandra

AU - Sugano, Yuya

AU - Wengi, Agnieszka

AU - Fisi, Viktoria

AU - Lindtoft Rosenbaek, Lena

AU - Mariniello, Marta

AU - Loffing-Cueni, Dominique

AU - McCormick, James A

AU - Fenton, Robert A

AU - Loffing, Johannes

N1 - © 2021 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.

PY - 2021/9

Y1 - 2021/9

N2 - 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.

AB - 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.

KW - NKCC2

KW - ion homeostasis

KW - kidney

KW - phosphorylation

KW - strain differences

KW - K-CL COTRANSPORTER

KW - SPAK

KW - PROTEIN

KW - SALT

KW - DISTAL CONVOLUTED TUBULE

KW - NACL COTRANSPORTER

KW - STRAIN DIFFERENCES

KW - DEOXYCORTICOSTERONE ACETATE

KW - SODIUM-CHLORIDE COTRANSPORTER

KW - THICK ASCENDING LIMB

U2 - 10.1111/apha.13705

DO - 10.1111/apha.13705

M3 - Journal article

C2 - 34114742

VL - 233

JO - Acta Physiologica (Print)

JF - Acta Physiologica (Print)

SN - 1748-1708

IS - 1

M1 - e13705

ER -