Objectives: Medial vascular calcification (MVC) is a complex process of arterial wall hardening, and the most common ectopic calcification associated to chronic kidney disease (CKD) (1,2). Our aim has been to study the very early structural and genomic changes that affect vascular cells and initiate the pathogenesis of MVC.
Methodology: We have used 5/6 nephrectomized rats fed with high dietary Pi (1,2%). Aortas were analyzed at different time points for Ca2+ deposition by colorimetry. Ca/P deposition was studied by Scanning Electron Microscopy (SEM), and with alizarin red and von Kossa stainings. Inorganic pyrophosphate (PPi) was measured by fluorimetry and gene expression was studied by Real Time PCR. Tissue-Nonspecific Alkaline Phosphatase (TNAP) activity in the artery wall was studied by NBT and fast blue RR salt staining. Several blood plasma parameter concentrations were also determined: urea, creatinine, Pi and Ca2+(colorimetry); PPi (fluorometry) and TNF-, PTH and FGF-23 (ELISA).
Findings: The earliest change detected in the aortic wall was an increase of aortic TNAP activity and mRNA expression 11 weeks after nephrectomy. Aortic Ca2+ rise and Ca/Pi deposition were only noticed after 12 weeks. Increased mRNA expressions of calcification-related genes (RUNX2, BMP2, Pit1, Pit2, HOXA10, PHOSPHO1, Fetuin-A, ANKH, OPN and ENPP1), were also found after TNAP changes. Increased plasma urea, creatinine, FGF-23, PTH and Pi were also noticed. Although we did not find changes in blood or aortic PPi, it is still possible that changes occur at later stages not studied in this work.
Significance: Not much is known about the early stages of MVC pathogenesis, which is mandatory to determine the order of events and the development of efficacious therapies. Our results underpin a role of TNAP as, most likely, the earliest player to trigger the calcification process. However, the TNAP specific role has still to be determined because PPi depletion has not been observed. The agents responsible for TNAP overexpression are still unkonwn, but the could involve, at least, uremic toxins that should be identified in order to provide the medical community with new pharmacological targets to treat and/or reverse this prevalent disease.