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Abstract: FR-PO614

Different NKCC2 Amino Acid Sequences Between 129/Sv and C57BL/6 Mice Affect Analysis of NKCC2 Phosphorylation with Phosphoform-Specific Antibodies

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Moser, Sandra, University of Zurich, Zurich, Switzerland
  • Loffing, Johannes, University of Zurich, Zurich, Switzerland
Background

The furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) of the thick ascending limb is critical for the renal control of electrolyte and fluid homeostasis. The activity of NKCC2 is regulated via phosphorylation of several serine and threonine (T) residues in the N-terminal tail of the co-transporter. To study NKCC2 function, phosphoform-specific antibodies directed against these phosphorylation sites (e.g. T96 and T101) have been developed and applied in studies on mouse models. The most frequently used mouse strains are 129Sv and C57BL/6 mice. Surprisingly, when we tried to detect phosphorylated NKCC2 (pNKKC2) with anti pT96/pT101 NKCC2 antibodies, we detected a strong pNKCC2 signal only in kidneys from 129Sv mice but not in kidney from C57BL/6 mice. In the latter, only some unspecific cross-reactivity of the pNKCC2 antibodies with the phosphorylated thiazide-sensitive NaCl cotransporter was seen.

Methods

To address this unexpected finding, we compared 129Sv and C57BL/6 mice via database analysis, metabolic cage experiments, quantitative RT-PCR and immunoblotting.

Results

Database analysis revealed that C57BL/6 mice have a five amino acid deletion (ΔT96-N100) in NKCC2, which lies in the region of the epitopes recognized by most anti-pNKCC2 antibodies. Although we observed strain differences in urinary Ca2+ and Mg2+ excretion and in the expression of several renal ion transporters and channels between 129Sv and C57BL/6 mice, these differences are likely not related to the five amino acid deletion in NKCC2. When we crossbred 129Sv and C57BL/6 mouse strains, mice of the F2 generation with the deletion (ΔT96-N100 mice) and mice without the deletion (control mice) were phenotypically similar. In particular, there were no differences in NKCC2 mRNA and protein abundances between the ΔT96-N100 and control mice. Nevertheless, pNKCC2 remained barely detectable in ΔT96-N100 mice using the antibodies against the full-length epitope.

Conclusion

Our study reports an important difference between the NKCC2 amino acid sequences of 129Sv and C57BL/6 mice that does not appear to significantly affect NKCC2 function, but strongly interferes with the detection of NKCC2 phosphorylation via phosphoform-specific antibodies. This needs to be considered when studying NKCC2 regulation in different mouse strains and when cross-breeding mice.