α1 Na,K-ATPase and Na,K,2Cl-Cotransporter/D3mit3 Loci Interact to Increase Susceptibility to Salt-Sensitive Hypertension in Dahl SHSD Rats

Abstract

Background: Essential (multigenic) hypertension is a complex multifactorial disease whose genetic etiology has not been unraveled on a major locus-effect investigative paradigm. As with other complex genetic diseases, applying an interacting loci paradigm could be critical in the elucidation of genetic determinants. Having defined the α1 Na,K-ATPase (α1NK) as a hypertension susceptibility gene in Dahl salt-sensitive (Dahl S) rats, we determined whether α1NK interacts with another renal epithelial Na transporter to increase susceptibility to salt-sensitive hypertension. We focused on α1NK and Na,K,2Cl-cotransporter (NKC) as an a priori candidate interacting gene pair because they comprise a functionally linked Na transport system in renal thick ascending limb of Henle (TALK) epithelial cells and exhibit altered function in prehypertensive Dahl S rats in contrast to Dahl salt-resistant normotensive (Dahl R) rats. Material and Method: Cosegregation analysis of α1NK and NKC loci was done in a (Dahl S × Dahl R) F2 cohort characterized for blood pressure by radiotelemetry using the D2mgh11 microsatellite marker in the α1NK gene and the D3mit3 microsatellite marker close to the NKC gene (NKC/D3mit3 locus). Single locus and digenic analyses were performed to establish the individual and interactive genetic contribution to salt-sensitive hypertension. Molecular analysis was then done to support the NKC gene as the likely candidate gene interacting with α1NK in Dahl salt-sensitive hypertension pathogenesis. Results: Compared with respective single locus analysis, digenic analysis of 96 F2 (Dahl S × Dahl R) hybrid male rats revealed cosegregation of α1NK and NKC/D3mit3 loci as interacting pair with salt-sensitive hypertension with markedly increased significance for systolic (one-way ANOVA p = 10-6), diastolic (p = 10-5), and mean arterial (p = 10-6) blood pressures. Concordantly, two-way ANOVA detected interaction between α1NK and NKC loci in determining the levels of systolic (p = 0.004), diastolic (p = 0.008), and mean arterial (p = 0.006) pressures. To unravel potential NKC molecular dysfunction(s) involved in hypertension pathogenesis, we investigated putative differences between Dahl S and Dahl R rats in nucleotide sequence and isoform gene expression of the renal-specific Na,K,2Cl-cotransporter. Molecular analysis revealed an inversion of alternatively spliced NKC-isoform ratios (4B:4A:4F) between Dahl S and Dahl R prehypertensive kidneys supported by four mutations in intron-3 immediately upstream to alternatively spliced exons 4B, 4A, and 4F. No nucleotide changes were detected within the aminoacid encoding exons of NKC. Conclusions: Altogether, these current data and previous characterization of the role of the Q276L α1NK molecular variant in Dahl S hypertension provide cumulative compelling evidence that α1NK and NKC/D3mit3 loci interact to increase susceptibility to hypertension in Dahl S rats and that NKC is the likely candidate gene that interacts with α1NK. More importantly, the data substantiate gene interaction as an operative mechanism in multigenic hypertension.