Background: Cardiovascular calcification is a significant cause of morbidity and mortality in patients with end-stage kidney disease (ESKD). Several metabolic and hormonal pathways have been implicated in renal failure-associated calcification. We used an in vitro system to explore the molecular pathway in renal failure associated aortic valve calcification.
Methods:Rat aortic valve interstitial cells (AVIC) were incubated with parathyroid hormone (PTH), adenine, TGF-β and 2 concentrations of phosphate: 3.5 mM (P3.5), which reflects mild to moderate renal failure, and 7 mM (P7), which indicates ESKD. Mineralization in AVIC was detected by von Kossa staining and calcium quantification. The osteoblastic proteins Runx-2 (early osteoblast marker), osteocalcin (late osteoblast marker) and osteopontin were examined by real-time PCR, western blot and immunostaining. To determine the function of phosphate in aortic valve calcification (AVC), cells were pretreated with foscarnet, a known inhibitor of phosphate cotransporter Pit-1, as well as silencing of Pit-1 using specific si-RNA.
Results: We confirmed that phosphate is the most efficient inducer of AVIC calcification in renal disease. P3.5 upregulated osteoblast genes after 7 days, but longer incubation with phosphate for 14 days decreased Runx-2 to control levels, increased osteocalcin and decreased osteopontin significantly less than the control. On the other hand, osteoblastic proteins were increased in P7 treated cells after 14 days.
Foscarnet inhibited phosphate-induced calcification, although Runx-2 expression was unchanged and osteocalcin level decreased.
However, foscarnet increased osteopontin expression after 7 and 14 days in P3.5 treated cells, while no change in expression was noted after 14 days in the P7 treated cells.
Silencing of Pit-1 did not affect calcification or osteoblastic genes expression.
Conclusions: Phosphate is a major determinant of AVC in patients with chronic renal failure. Phosphate-induced calcification involves several molecular pathways at various stages of renal impairment. Furthermore, the effects of phosphate on calcification are Pit-1 independent.
