Inhibiting the phosphatase activity of soluble epoxide hydrolase attenuates chronic kidney disease-induced calcific aortic valve disease in rats

Abstract

Calcific aortic valve disease (CAVD) is the most frequent valvulopathy ultimaltely leading to aortic stenosis, one of the driving causes of an increased morbidity and mortality in aging and in patients with chronic kidney disease (CKD). Up to date, there is no available pharmacological treatment to prevent the development and progression of CAVD. Previous studies suggested that the bifunctional enzyme, soluble epoxide hydrolase (sEH), is involved in the regulation of cardiovascular calcification development. Our study aimed to investigate whether the phosphatase activity of sEH is involved in CAVD. Sprague Dawley sEH-P knocking rats (sEH-P KI) were generated using CRISPR/Cas9 method. Eighteen-week-old WT and sEH-P KI rats underwent subtotal nephrectomy (5/6Nx) to mimic CKD. These animals were afterword fed with a high-phosphate chow diet (1.8% phosphorus). In parallel, sham-operated animals on a standard diet (0.9% total phosphorus) served as controls. Twelve weeks after surgery, renal and cardiac parameters were determined and histological analysis of aortic valves was performed. Twelve weeks after surgery, the development of renal dysfunction in 5/6Nx + phosphate WT rats was shown by the augmentation in plasma creatinine and urea nitrogen concentrations and the decline in estimated glomerular filtration rate compared to sham-operated WT rats. These animals also displayed anemia, polyuria and polydipsia. The genetic inhibition of sEH-P did not allow to prevent these alterations. However, inhibiting sEH-P limited hydroxyapatite crystals deposition in the aortic valve of 5/6Nx + phosphate rats compared to 5/6Nx + phosphate WT rats (Fig. 1B, C), associated with a prevention of the decrease in intercusp distance and of the increase in aortic valve mean pressure gradient and in peak aortic valve velocity (Fig. 1A). In addition, left-ventricular diastolic (E/e′) and systolic dysfunction (fractional shortening) as well as cardiac fibrosis were prevented by sEH-P inhibition. Additional experiments on recombinant human and rat sEH allowed to demonstrate that sEH-P metabolizes pyrophosphate anions (PPi), a potent inhibitor of cardiovascular calcification, and that PPi degradation is efficiently prevented by sEH-P inhibition. Taking altogether, these results demonstrate that inhibition of sEH-P prevents aortic valve calcification and its hemodynamical consequences in the 5/6Nx + phosphate rat model, and may serve as a potential therapeutic strategy for the treatment of CAVD.