MO550: Vascular Cells Senescence and Modifications in NRF2 Pathway in a Model of URaEMIC Vascular Calcification
Authors: Laget Jonas , Hobson Sam , Muyor Karen , Duranton Flore , Cortijo Irene , Bartochowski Piotr , Jover Bernard , Dominique Lajoix Anne , Stenvinkel Peter , Argiles Ciscart Angel , Kublickiene Karolina , Gayrard Nathalie 
Published: May 2022

Abstract

BACKGROUND AND AIMS
Vascular dysfunction including vascular calcification (VC), is a consequence of ageing and chronic kidney disease (CKD) that increase the risk of cardiovascular events. Accumulation of senescent cells in arteries can lead to structural and functional alterations and contributes to increased arterial stiffness, reduced compliance and impaired contractility. We tested the hypothesis that senescent cells in the arterial wall are involved in the pathophysiology of vascular calcification in a rat model of CKD and VC induced by a high phosphate diet and vitamin D supplementation.

METHOD
Eight-week-old Sprague–Dawley rats underwent subtotal nephrectomy (SNx, n = 18), or no surgery (Control, n = 6). Eight weeks after, control animals (n = 6) and part of the SNx group (n = 6) were maintained on the standard diet while the remaining rats underwent a 4-week VC-diet, where a standard diet was supplemented with high phosphate and 1-hydroxy-vitamin D at a dose of 0.1 µg/day (SNx 0.1 group, n = 6) or 0.4 µg/day (SNx 0.4 group, n = 6). At the end of the protocol, renal function, blood pressure and VC (von Kossa) were studied. mRNA and protein expressions of cellular senescence markers (p16, p21, yH2AX), antioxidants (NRF2, Nqo-1), VC inducers/inhibitors and osteoblastic transition drivers were assessed by qPCR, immunohistochemistry (IHC), or immunofluorescence (IF). Correlations between mRNA or protein levels and VC were computed for the VC-diets animals (n = 12).

RESULTS
A decrease in creatinine clearance (P &lt; 0.05), increase in urinary protein excretion (P &lt; 0.05) and tubulointerstitial fibrosis were observed in the three SNx groups. VC-diets induced vascular calcification in thoracic aorta and aortic arch associated with an increase in pulse pressure (P &lt; 0.01). NRF2 mRNA and protein expression increased in the calcified groups (P &lt; 0.05) and were positively correlated with VC (Figure 1). In addition, mRNA expression of NRF2 target Nqo-1 was strongly correlated to VC (P &lt; 0.01). p16 mRNA (P &lt; 0.05) and protein (IHC, P &lt; 0.01) levels were correlated to VC; a similar expression pattern was observed for p21 protein (P &lt; 0.05) (Figure 1). The number of yH2AX positive cells increased in calcified groups (IF, P &lt; 0.05) and some of the positive cells for p16 were co-labelled with yH2AX (Figure 1).

CONCLUSION
In our rat model of induced VC associated with CKD, NRF2 and downstream targets expression was increased in thoracic aorta, confirming the link between NRF2 and VC. This might be a response mechanism as observed with other ‘protective’ factors with increased mRNA expression in this model (data not presented here). Furthermore, we observed that cellular senescence was clearly associated with VC. The co-labelling for p16 and yH2AX in some vascular cells in the media layer, exclusively observed in calcified aortas, is another clue that highlights the link between VC and vascular cell senescence.