α1 Na,Κ-ATPase and Na,Κ,2 Cl-cotransporter/D3mit3 loci interact to increase susceptibility to salt-sensitive hypertension in Dahl SHSD rats

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 alpha1 Na,K-ATPase (alpha 1NK) as a hypertension susceptibility gene in Dahl salt-sensitive (Dahl S) rats, we determined whether alpha 1NK interacts with another renal epithelial Na transporter to increase susceptibility to salt-sensitive hypertension. We focused on alpha 1NK 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 (TALH) 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 alpha 1NK and NKC loci was done in a (Dahl S x Dahl R) F2 cohort characterized for blood pressure by radiotelemetry using the D2mgh11 microsatellite marker in the alpha 1NK 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 alpha 1NK in Dahl salt-sensitive hypertension pathogenesis. Results: Compared with respective single locus analysis, digenic analysis of 96 F2 (Dahl S x Dahl R) hybrid male rats revealed cosegregation of alpha 1NK 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 alpha 1NK 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 alpha 1NK molecular variant in Dahl S hypertension provide cumulative compelling evidence that alpha 1NK 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 cu 1NK. More importantly, the data substantiate gene interaction as an operative mechanism in multigenic hypertension.