alpha 1 Na,Kappa-ATPase and Na,Kappa,2 Cl-cotransporter/D3mit3 loci interact to increase susceptibility to salt-sensitive hypertension in Dahl S-HSD rats

Background: Essential (multigenic) hypertension is a complex multifactorial disease whose genetic etiology has not been unraveled on a major locus-eff ect investigative paradigm. As with other complex genetic diseases, applyin g an interacting loci paradigm could be critical in the elucidation of gene tic determinants. Having defined the alpha1 Na,K-ATPase (alpha 1NK) as a hy pertension susceptibility gene in Dahl salt-sensitive (Dahl S) rats, we det ermined whether alpha 1NK interacts with another renal epithelial Na transp orter to increase susceptibility to salt-sensitive hypertension. We focused on alpha 1NK and Na,K,2Cl-cotransporter (NKC) as an a priori candidate int eracting 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 d one in a (Dahl S x Dahl R) F2 cohort characterized for blood pressure by ra diotelemetry using the D2mgh11 microsatellite marker in the alpha 1NK gene and the D3mit3 microsatellite marker close to the NKC gene (NKC/D3mit3 locu s). Single locus and digenic analyses were performed to establish the indiv idual and interactive genetic contribution to salt-sensitive hypertension. Molecular analysis was then done to support the NKC gene as the likely cand idate gene interacting with alpha 1NK in Dahl salt-sensitive hypertension p athogenesis. Results: Compared with respective single locus analysis, digenic analysis o f 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 hypertensio n 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 = 0.00 8), and mean arterial (p = 0.006) pressures. To unravel potential NKC molec ular 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. M olecular analysis revealed an inversion of alternatively spliced NKC-isofor m ratios (4B:4A:4F) between Dahl S and Dahl R prehypertensive kidneys suppo rted by four mutations in intron-3 immediately upstream to alternatively sp liced 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 o f the role of the Q276L alpha 1NK molecular variant in Dahl S hypertension provide cumulative compelling evidence that alpha 1NK and NKC/D3mit3 loci i nteract to increase susceptibility to hypertension in Dahl S rats and that NKC is the likely candidate gene that interacts with cu 1NK. More important ly, the data substantiate gene interaction as an operative mechanism in mul tigenic hypertension.