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.

Sex-specific adaptations to high-salt diet preserve electrolyte homeostasis with distinct sodium transporter profiles

“…TAL NKCC2pS87 was also 50% lower in F than M on either diet (P = 0.016 on NS, P = 0.002 on HS), which suggests less NKCC2 transport activity along the TAL in F. We did not previously detect this sex difference with an antibody directed to NKCC2pT96T101 (18). This discrepancy is now explained by a recent report of a five amino acid deletion (DeltaF97-T101) in NKCC2 in the C57BL/6 strain that prevents detection of NKCC2 phosphorylation at this site; the site at NKCC2pS87 is not mutated (55). The nonspecific signals previously obtained with antibodies to NKCC2pT96T101 in the C57BL/6 strain can be attributed to cross-reaction with a homologous phosphorylation site on the Na þ -Cl À cotransporter (55).…”

“…This discrepancy is now explained by a recent report of a five amino acid deletion (DeltaF97-T101) in NKCC2 in the C57BL/6 strain that prevents detection of NKCC2 phosphorylation at this site; the site at NKCC2pS87 is not mutated (55). The nonspecific signals previously obtained with antibodies to NKCC2pT96T101 in the C57BL/6 strain can be attributed to cross-reaction with a homologous phosphorylation site on the Na þ -Cl À cotransporter (55). Lower NHE3, cldn-2, and NKCC2p in females would likely facilitate more rapid adaptation to a dietary Na þ challenge by expediting natriuresis and diuresis along the proximal nephron.…”

Profiling renal sodium transporters in mice with nephron Ift88 disruption: Association with sex, cysts, and blood pressure

“…There were no significant differences in the other Na + transporters/channels between high salt fed male Ift88 KO 2 months post‐DOX and age‐ and diet‐matched control male mice (Figures 2 and 3). Note that phosphorylated (T 96/101 ) NKCC2 was not measured due to a recent report demonstrating that currently available anti‐phosphorylated (T 96/101 ) NKCC2 antibodies cross react with phosphorylated NCC specifically in C57BL/6 mice (Moser et al, 2021).…”

Assessment of NKCC2 phosphorylation in mice

“…In this issue of Acta Physiologica, Moser et al report experiments assessing the specificity of commonly used phosphospecific NKCC2 antibodies in mice. 1 These antibodies, which target the phosphorylation sites at threonine 96 and 101 of the NKCC2 protein, are known to be notoriously unreliable. In an attempt to address this issue, the authors used genomic sequence analysis to reveal that NKCC2 of the commonly used C57BL/6 mouse strain has a short 5-amino acid deletion from amino acids 97-101.…”

“…In this issue of Acta Physiologica , Moser et al report experiments assessing the specificity of commonly used phospho‐specific NKCC2 antibodies in mice 1 . These antibodies, which target the phosphorylation sites at threonine 96 and 101 of the NKCC2 protein, are known to be notoriously unreliable.…”