Calcification is associated with loss of functional calcium-sensing receptor in vascular smooth muscle cells.
OBJECTIVE
Vascular calcification (VC) is highly correlated with increased morbidity and mortality in advanced chronic kidney disease (CKD) patients. Allosteric modulation of the calcium-sensing receptor (CaR) by calcimimetics inhibits VC in animal models of advanced CKD. Here, we investigated the expression of the CaR in the vasculature and tested the ability of calcimimetics to prevent vascular smooth muscle cell (VSMC) calcification in vitro.


RESULTS
Immunohistochemical staining demonstrated that CaR protein is present in VSMC in normal, non-calcified human arteries. In contrast, low levels of CaR immunoreactivity were detected in atherosclerotic, calcified arteries. Immunfluorescence and immunoblotting revealed that CaR protein was also expressed by human and bovine VSMC in vitro. Acute stimulation of VSMC with increased Ca2+ stimulated extracellular signal-regulated kinase (ERK1/2) phosphorylation, suggesting that the VSMC CaR is functional. VSMC CaR expression decreased when these cells deposited a mineralized matrix or following 24 h incubation in mineralization medium with increased (i.e. 1.8 or 2.5 mM) Ca2+. Culturing VSMC in mineralization medium containing 1.8 and 2.5 mM Ca2+ or with the membrane-impermeant CaR agonist Gd3+ enhanced mineral deposition compared with that observed in 1.2 mM Ca2+. Over-expression of dominant-negative (R185Q) CaR enhanced, whereas the calcimimetic R-568 attenuated, VSMC mineral deposition.


CONCLUSIONS
These results demonstrate that: (i) VSMCs express a functional CaR; (ii) a reduction in CaR expression is associated with increased mineralization in vivo and in vitro; (iii) calcimimetics decrease mineral deposition by VSMC. These data suggest that calcimimetics may inhibit the development of VC in CKD patients.
Alam
Masih-ul
MU
Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Michael Smith Building, Oxford Road, Manchester M139PT, UK.
Kirton
John Paul
JP
Wilkinson
Fiona L
FL
Towers
Emily
E
Sinha
Smeeta
S
Rouhi
Mansour
M
Vizard
Thomas N
TN
Sage
Andrew P
AP
Martin
David
D
Ward
Donald T
DT
Alexander
Marie Yvonne
MY
Riccardi
Daniela
D
Canfield
Ann E
AE
D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D002114:Calcinosis / Q000209:etiology*; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002908:Chronic Disease; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005682:Gadolinium / Q000494:pharmacology; D006801:Humans; D007674:Kidney Diseases / Q000150:complications; D008903:Minerals / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D010627:Phenethylamines; D010766:Phosphorylation; D011437:Propylamines; D044169:Receptors, Calcium-Sensing / Q000032:analysis / Q000502:physiology*
D000814:Aniline Compounds; D008903:Minerals; C107873:N-(2-chlorophenylpropyl)-1-(3-methoxyphenyl)ethylamine; D010627:Phenethylamines; D011437:Propylamines; D044169:Receptors, Calcium-Sensing; D005682:Gadolinium; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium