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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  • Sandra Moser, University of Zurich
  • ,
  • Yuya Sugano, University of Zurich
  • ,
  • Agnieszka Wengi, University of Zurich
  • ,
  • Viktoria Fisi, University of Zurich
  • ,
  • Lena Lindtoft Rosenbaek
  • Marta Mariniello, University of Zurich
  • ,
  • Dominique Loffing-Cueni, University of Zurich
  • ,
  • James A McCormick, Oregon Health & Science University, Portland, OR, USA
  • ,
  • Robert A Fenton
  • Johannes Loffing, University of Zurich

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.

OriginalsprogEngelsk
Artikelnummere13705
TidsskriftActa Physiologica (Print)
Vol/bind233
Nummer1
Antal sider16
ISSN1748-1708
DOI
StatusUdgivet - sep. 2021

Bibliografisk note

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

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