MO056MODIFICATION OF P16, P21 AND NRF2 EXPRESSION IN A MODEL OF UREMIC VASCULAR CALCIFICATION

Abstract

Abstract Background and Aims Vascular calcification (VC) is a consequence of ageing that confers development of future cardiovascular events. Accumulation of senescent cells can lead to structural and functional abnormalities in vessel wall towards increased stiffness, reduced compliance and impaired contractile and dilatory capacity. Thus, accumulation of senescent cells in the arterial wall could also contribute to the pathophysiology of VC. To test this hypothesis, the presence of cellular markers of senescence, p16, p21 and NRF2, was assessed in aorta from rat model of VC associated with chronic kidney disease (CKD). Method Six-week-old Sprague-Dawley rats underwent 5/6th subtotal nephrectomy (SNx, n=6) or no surgery (Control, n=1). After 8 weeks of renal failure, the regular chow was supplemented with high phosphate (1.2%) and vitamin D (1 µg/day, 5 days per week) for 1 or 4 weeks to initiate vascular calcification (SNx-VC group). At the end of the protocol (Figure 1), blood pressure was measured, and thoracic aorta was taken for determination of calcification by Von Kossa staining, and protein and gene expressions of p16, p21, and NRF2 by immunostaining and qPCR, respectively. Results After 4 weeks of dietary intervention, SNx rats showed an increase in pulse pressure (88±15mmHg vs 35 mmHg for the control). Marked VC was also observed in these animals, 17% of calcified area vs <1 % in the control rat. Calcification was focal giving strong and no calcified areas on the same aorta. Expressions of p16, p21and NRF2 proteins were enhanced at the site of calcification in the SNx-VC 4-week (4W) group whereas it was unchanged in aortic media without calcification compared to the control rat (Figure 2). In SNx-VC 1-week (1W) rats, no change in pulse pressure or vascular calcification was observed without obvious changes for staining patterns of selected markers. Using qPCR we found p16 gene expression to be higher in the most calcified aortas (4W) (fold-change = 4.32 vs 1W) and p21 gene expression to be slightly increased (fold-change = 0.53 vs 1W). NRF2 gene expression was enhanced in 1W group compared to control, but decreased in the most calcified samples (4W). Conclusion This pilot study suggests that uraemia-induced cellular senescence accompanies VCs, as suggested by the modified expression of p16 or NRF2 genes. Our observations deserve exploration in larger studies using additional senescence markers for validation. If so, cellular senescence kinetics will be evaluated in order to test whether senolytics compounds could be a therapeutic option to arrest VC in CKD.