Unique situations in female physiology require volume retention. Accordingly, a dimorphic regulation of the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) has been reported, with a higher activity in females than in males. However, little is known about the hormones and mechanisms involved. Here, we present evidence that estrogens, progesterone, and prolactin stimulate NCC expression and phosphorylation. The sex difference in NCC abundance, however, is species dependent. In rats, NCC phosphorylation is higher in females than in males, while in mice both NCC expression and phosphorylation is higher in females, and this is associated with increased expression and phosphorylation of full-length STE-20 proline-alanine-rich kinase (SPAK). Higher expression/phosphorylation of NCC was corroborated in humans by urinary exosome analysis. Ovariectomy in rats resulted in decreased expression and phosphorylation of the cotransporter and promoted the shift of SPAK isoforms toward the short inhibitory variant SPAK2. Conversely, estradiol or progesterone administration to ovariectomized rats restored NCC phosphorylation levels and shifted SPAK expression and phosphorylation towards the full-length isoform. Estradiol administration to male rats induced a significant increase in NCC phosphorylation. NCC is also modulated by prolactin. Administration of this peptide hormone to male rats induced increased phosphorylation of NCC, an effect that was observed even using the ex vivo kidney perfusion strategy. Our results indicate that estradiol, progesterone, and prolactin, the hormones that are involved in sexual cycle, pregnancy and lactation, upregulate the activity of NCC.

BackgroundMutations in four genes, WNK lysine deficient protein kinase 1 and 4 (WNK1 and WNK4), kelch like family member 3 (KLHL3), or Cullin 3 (CUL3), can result in familial hyperkalemic hypertension (FHHt), a rare Mendelian form of human arterial hypertension. Although all mutations result in an increased abundance of WNK1 or WNK4, all FHHt-causing CUL3 mutations, resulting in the skipping of exon 9, lead to a more severe phenotype.MethodsWe created and compared two mouse models, one expressing the mutant Cul3 protein ubiquitously (pgk-Cul3∆9) and the other specifically in vascular smooth muscle cells (SM22-Cul3∆9). We conducted pharmacologic investigations on isolated aortas and generated stable and inducible HEK293 cell lines that overexpress the wild-type Cul3 or mutant Cul3 (Cul3∆9) protein.ResultsAs expected, pgk-Cul3∆9 mice showed marked hypertension with significant hyperkalemia, hyperchloremia and low renin. BP increased significantly in SM22-Cul3∆9 mice, independent of any measurable effect on renal transport. Only pgk-Cul3∆9 mice displayed increased expression of the sodium chloride cotransporter and phosphorylation by the WNK-SPAK kinases. Both models showed altered reactivity of isolated aortas to phenylephrine and acetylcholine, as well as marked acute BP sensitivity to the calcium channel blocker amlodipine. Aortas from SM22-Cul3∆9 mice showed increased expression of RhoA, a key molecule involved in regulation of vascular tone, compared with aortas from control mice. We also observed increased RhoA abundance and t1/2 in Cul3∆9-expressing cells, caused by decreased ubiquitination.ConclusionsMutations in Cul3 cause severe hypertension by affecting both renal and vascular function, the latter being associated with activation of RhoA.