MO454: Increased Senescent Cell Burden and Alterations in NRF2 Pathway Drive Uraemic Vascular Calcification in Humans

Abstract BACKGROUND AND AIMS Vascular calcification is a clinical sequelae of chronic kidney disease (CKD) that is associated with high cardiovascular-related mortality in end-stage kidney failure (ESKF) patients. Increased senescent cell burden and dysregulation of the NRF2 pathway, the master regulator of antioxidant genes, have been suggested to play an important role in the onset of multiple non-communicable burden-of-lifestyle diseases, including but not limited to diabetes, obesity and CKD. Thus, we investigated if senescence and NRF2 pathways may serve as drivers of uraemia-induced vascular calcification in human arteries. METHOD ESKF patients without epigastric artery calcification and coronary artery calcification (CAC) (n = 8) were matched with patients with severely calcified epigastric arteries and the presence of CAC (n = 8). In all subjects, samples of epigastric arteries were obtained during living donor kidney transplantation surgery. Gene (qPCR) and protein expression (immunohistochemistry) of cellular senescence markers (p16, p21), NRF2 pathway candidates (NRF2, NQO1, CAT, SOD1) and calcification markers (RUNX2, ALPL) were assessed. In addition, four groups of aortic vascular smooth muscle cells (VSMCs) were treated as follows: i) control media; ii) osteogenic media (Pi = 2.5mM); iii) osteogenic media + pooled serum from non-ESKD subjects; or iv) osteogenic media + pooled uraemic serum from ESKF patients that had severely calcified epigastric arteries (n = 8). Calcification in cell culture experiments was assessed using BoneTag Optical Probe and normalized for protein content, while gene expression analysis for a selection of calcification, senescence and NRF2-related markers, and SA-beta-GAL staining, were performed (n = 4 each). RESULTS At the gene expression level, severely calcified epigastric arteries as assessed by von Kossa staining (Figure 1a) had preserved ALPL expression but significantly increased RUNX2 expression (P < 0.01; Figure 1b) when compared with non-calcified vessels. Gene expression of p16, but not p21, was increased in the calcified compared with non-calcified group (P < 0.001, Figure 1c), while both p16 and p21 protein expressions were elevated in the media layer of calcified vessels (Figure 1c). NRF2 and downstream target gene expressions remained similar between the two groups, however, NRF2 protein expression was increased in the non-calcified group. In cell culture studies, uraemic serum-induced VSMC calcification was increased when compared to non-uraemic serum and osteogenic controls (P < 0.0001, Figure 2a). This was further accompanied by increased ALPL gene expression (P < 0.0001, Figure 2b) and reduced NRF2, SOD1 and NQO1 gene expression (all P < 0.01, Figure 2c). SA-beta-gal staining and p16/p21 gene expression analysis revealed that uraemic serum-induced calcification was not associated with increased senescent cell burden. CONCLUSION Established calcification in ESKF patients is associated with increased senescent cell burden, but preserved NRF2 pathway markers, in epigastric arteries. In contrast, induced calcification was accompanied by the diminished activity of the NRF2 pathway, but not senescence pattern, using an in vitro approach in VSMCs. Our translational data indicate that established and induced calcification may reflect different pathophysiological processes, supporting data from a parallel study using 5/6 rats (Laget, Hobson et al., unpublished data). Future studies should consider the timing of initiation of NRF2 agonist/senolytic treatment when targeting calcification in ESKF patients.