Loss of Clcn6 Alters Expression of Nearby Regulatory Blood Pressure Genes but Does Not Affect High Salt Induced Mortality in Dahl Salt‐Sensitive Rats

Abstract

Genome‐wide association studies in both humans and Dahl salt‐sensitive (SS) rats have identified the AGTRAP‐PLOD1 locus gene cluster as an area of interest that contains several genes with the ability to regulate blood pressure, including CLCN6, MTHFR, NPPA,and NPPB. We previously demonstrated that a functional knock‐out of Clcn6 on the Dahl SS rat background (SS‐Clcn6) had significantly reduced diastolic blood pressure (BP) compared to WT rats when challenged with a high 4% NaCl (HS) diet to induce hypertension. Increased stroke risk from high blood pressure is well established, and genetic variants, such as those in CLCN6 that reduce BP may also be expected to lower the risk of stroke within this population. Furthermore, a recent paper reported that loss of function mutations in CLCN6 were associated with long‐term survival in a small cohort of sickle cell anemia patients with lower stroke rates. We hypothesized that loss of Clcn6 would delay HS‐induced mortality of Dahl SS rats on a 8% NaCl diet by slowing or preventing lethal cerebrovascular events. However, we observed no significant differences in longevity or neuronal cell death between the SS‐Clcn6 knock‐out and SS‐WT groups. Further assessment led us to examine whether a mutation in Clcn6 could alter mRNA expression of nearby Mthfr, Nppa, and Nppb genes, thereby complicating the genetic phenotype of our knockout animal. We found that on normal salt (NS) diets, renal Mthfr mRNA and protein expression were significantly increased in the SS‐Clcn6 rats, while no differences were observed between WT and knockout rats on a HS diet. MTHFR encodes Methylene tetrahydrofolate reductase (MTHFR), which is involved in reducing homocysteine to methionine, and elevated levels of circulating homocysteine (tHcy) are associated with higher cardiovascular and thromboembolic risk. No significant differences in circulating tHCy between SS‐WT and SS‐Clcn6 (10.63 ± 0.47 vs 10.05 ± 1.12 nM/mL, respectively), were observed on a NS diet; however, there was a trend toward decreased tHcy in SS‐Clcn6 rats on a HS diet (11.85 ± 2.55 vs 6.90 ± 0.40 nM/mL, p=0.08). Genetic ablation of Clcn6 resulted in significant differences in NppamRNA levels in cardiac tissue from SS‐WT and SS‐Clcn6 rat in both normotensive (153.0 ± 23.3 vs 17.61 ±10.67 a.u., p<0.01) and hypertensive (614.9 ± 115.2 vs 153.0 ± 23.3 a.u., p<0.01) conditions. A substantial reduction in NppbmRNA expression in SS‐Clcn6 rats on a NS diet (66.04 ± 4.50 vs 24.01 ± 8.80 a.u., p<0.01) was also found. The observed genetc variances are controversial; while increased Mthfrexpression would be predicted to be protective, reduced Nppaand Nppbexpression could potentially be deleterious and prevent increased vasodilation, natriuresis, and diuresis that should normally occur to compensate for BP increases. Due to the similarity of this genetic locus in humans and rats, more studies are warranted to fully understand how genetic mutations in this cluster may be altering the expression of their neighbors as well as contributing to BP homeostasis.