Dysregulation of the WNK4-SPAK/OxSR1 pathway has a mild effect on NKCC2 activity

Background: The furosemide-sensitive Na + -K+ - 2Cl − cotransporter (NKCC2) reabsorbs 20% of filtered Na+ along the thick ascending limb (TAL), while and thiazide-sensitive NaCl cotransporter (NCC) reabsorbs 5-10% along the distal convoluted tubule (DCT). The WNK4-SPAK/OxSR1 pathway mediates NKCC2 phosphorylation at threonines 96 and 101 (pT96/pT101) and NCC phosphorylation at threonine 53 (pT53). This activates both cotransporters. A recent study reported that the commonly used pT96/pT101 pNKCC2 antibody cross-reacts with pT53 NCC in mice on the C57BL/6 background due to a 5 amino acid deletion, calling into question some previous findings regarding WNK4-SPAK/OxSR1 regulation of NKCC2. Although WNK4 is highly expressed along both TAL and DCT, dysregulation of WNK4-SPAK/OxSR1 pathway specifically causes Familial Hyperkalemic Hypertension (FHHt), which is exquisitely sensitive to thiazide diuretics, and thus a disease of NCC dysregulation. Using a new pNKCC2 antibody specific in C57BL/6 mice, we tested the hypothesis that the WNK4-SPAK/OxSR1 pathway more strongly activates NCC than it does NKCC2. Methods: A new pT96 NKCC2 antibody was validated in C57BL/6, 129Sv, and Slc12a3 −/− mice. The abundances of pT96 NKCC2 and pT53 NCC were evaluated in Wnk4 −/− , Oxsr1 −/− , Spak −/− , Oxsr1 −/− / Spak −/− mice, and several FHHt model, Cul3 +/−/Δ9 , Klhl3 −/− , and Klhl3 R528H/R528H mice. NKCC2 activity was confirmed by furosemide response test with thiazide pre-treatment in Klhl3 −/− mice. Both male and female were used and all genetically modified strains were on the C57BL/6 background. Results: The pT96 NKCC2 antibody detected pNKCC2 in C57BL/6 mice but not 129Sv mice, and did not cross-react with phosphorylated NCC. pT53 NCC was almost absent but pT96 NKCC2 was only slightly lower in Wnk4 −/− mice. pT53 NCC was almost absent with Spak deletion ( Spak −/− and Oxsr1 −/− / Spak −/− mice) but pT96 NKCC2 abundance did not differ from controls. Oxsr1 deletion ( Oxsr1 −/− and Oxsr1 −/− / Spak −/− mice) led to a modest lowering of pT96 NKCC2/total NKCC2 ratio but not pT96 NKCC2 abundance. Interestingly, immunofluorescence revealed that WNK4 expression was increased along not only DCT but also cortical TAL in Klhl3 −/− mice, but pT96 NKCC2 abundance was not changed. pT53 NCC abundance, but not pT96 NKCC2 abundance, was higher in two other FHHt mouse models with higher WNK4 ( Cul3 +/−/Δ9 and Klhl3 R528H/R528H mice). Consistent with no difference in pT96 NKCC2 abundance, urine Na + excretion after furosemide injection following thiazide treatment was similar between Klhl3 −/− and control mice. Conclusions: NCC is strongly phosphorylated by the WNK4-SPAK pathway, whereas NKCC2 is only mildly phosphorylated by the WNK4-OxSR1 pathway, suggesting another kinase can phosphorylate NKCC2. In FHHt models with Cul3 or Klhl3 mutations, NKCC2 phosphorylation is unchanged despite higher WNK4 abundance, explaining the thiazide-sensitivity of FHHt. Y.M. received a postdoctoral award from the Uehara Foundation; J.A.M. is funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK098141; M.C.B. is funded by CONACyT, Mexico, Grant 101720. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.