Overexpression of the sodium chloride cotransporter is not sufficient to cause familial hyperkalemic hypertension

Abstract

The sodium chloride cotransporter (NCC) is the primary target of thiazides diuretics, drugs used commonly for long-term hypertension therapy. Thiazides also completely reverse the signs of familial hyperkalemic hypertension (FHHt), suggesting that the primary defect in FHHt is increased NCC activity. To test whether increased NCC abundance alone is sufficient to generate the FHHt phenotype, we generated NCC transgenic mice; surprisingly, these mice did not display an FHHt-like phenotype. Systolic blood pressures of NCC transgenic mice did not differ from those of wild-type mice, even after dietary salt loading. NCC transgenic mice also did not display hyperkalemia or hypercalciuria, even when challenged with dietary electrolyte manipulation. Administration of fludrocortisone to NCC transgenic mice, to stimulate NCC, resulted in an increase in systolic blood pressure equivalent to that of wild-type mice (approximately 20 mm Hg). Although total NCC abundance was increased in the transgenic animals, phosphorylated (activated) NCC was not, suggesting that the defect in FHHt involves either activation of ion transport pathways other than NCC, or else direct activation of NCC, in addition to an increase in NCC abundance.

Figure 1. Generation of NCC transgenic mice

(A) BAC clone RP-24-263E2 was modified by recombineering in E. coli, to remove Herpud1 and the region of Nup43 extending to its 3′ untranslated region (UTR). The resulting NCC transgene contains 12.5kb of sequence upstream of the NCC transcription start, and the 3′ UTR of NCC (vector sequence is not shown). (B) Semi-quantitative PCR on genomic DNA extracted from the tails of potential Founders identified lines 727 and 743 as containing >12 and 5 copies of the NCC transgene; amplification of the β-globin gene confirmed equal template input.

Figure 2. NCC transgenic mice display increased total NCC protein expression

(A) Western blot analysis of whole kidney extracts from wild type (WT) and NCC transgenic (NCC-TG) mice was performed using antibodies against NCC and β-actin. (B) Densitometric quantitation was performed normalizing to β-actin, and expression of total NCC in NCC-TG (filled bars) relative to WT (open bars) mice was calculated. Wild type (WT), n=13; NCC transgenic (NCC-TG), n=16; values ± S.E.M., *p = 0.005. (C) Immunofluorescence showed that NCC expression is restricted to the DCT in NCC-TG mice.

Figure 3. NCC phosphorylation and cellular distribution are not altered in NCC-TG mice

(A) Western blot analysis of whole kidney extracts from wild type (WT) and NCC transgenic (NCC-TG) mice was performed using antibodies against phospho-NCC (T53) and β-actin. (B) Densitometric quantitation was performed normalizing to β-actin, and expression of phospho-NCC in NCC-TG (filled bars) relative to WT (open bars) mice was calculated. n=12 for each group; values ± S.E.M. (C) Immunofluorescence showed that in both WT and NCC-TG mice, total and phospho-NCC displayed expression at the apical membrane of the DCT.

Figure 4. NCC transgenic mice are normotensive

Systolic blood pressure was measured in wild type (open bars) and NCC transgenic mice (filled bars) after 2 weeks on standard (0.49% NaCl) and high (8% NaCl) salt diets, and 2 weeks of high salt diet with fludrocortisone in drinking water (17mg/l). There were no significant differences in systolic blood pressure on standard or high salt diet. Fludrocortisone treatment led to a similar, significant increase in systolic blood pressure in both wild type and NCC transgenic mice.

Figure 5. Normal electrolyte homeostasis in NCC transgenic mice

(A) Plasma K⁺ and (B) plasma Na⁺ did not differ between wild type (open bars) and NCC transgenic mice (filled bars) on standard (0.8% K⁺) or high potassium (5% K⁺) diets. Values are means ± S.E.M., n=19–22. (C) Urinary calcium:creatinine, measured from spot urine collections, did not differ between wild type (open bars) and NCC transgenic mice (filled bars) on standard (0.49% NaCl) or high (8%) NaCl diets. Values are means ± S.E.M., n=18–22.