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 (i) to elucidate these inconsistencies and (ii) 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 (i.e. 129Sv and in C57BL/6 mice). Moreover, a new phosphoform-specific mouse NKCC2 antibody was developed and characterized. RESULTS: Amino acid sequence alignment revealed that C57BL/6 mice have a strain-specific five amino acid 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 acid sequence of mouse NKCC2 that needs to be considered when analyzing NKCC2 phosphorylation in mice. The new pNKCC2 antibody circumvents this technical caveat. This article is protected by copyright. All rights reserved.
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 (i) to elucidate these inconsistencies and (ii) 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 (i.e. 129Sv and in C57BL/6 mice). Moreover, a new phosphoform-specific mouseNKCC2 antibody was developed and characterized. RESULTS: Amino acid sequence alignment revealed that C57BL/6 mice have a strain-specific five amino acid 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 pT96NKCC2 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 acid sequence of mouseNKCC2 that needs to be considered when analyzing NKCC2 phosphorylation in mice. The new pNKCC2 antibody circumvents this technical caveat. This article is protected by copyright. All rights reserved.
Entities:
Keywords:
Ion homeostasis; NKCC2; kidney; phosphorylation; strain differences
Authors: Diana L Torres-Pinzon; Donna L Ralph; Luciana C Veiras; Alicia A McDonough Journal: Am J Physiol Cell Physiol Date: 2021-10-06 Impact factor: 4.249