Local renin-angiotensin system is associated with bone mineral density of glucocorticoid-induced osteoporosis patients

Abstract

This study provides evidence that the role of local RAS is related to BMD in GIOP patients, and suggests that local RAS might influence RANKL/OPG signaling to modulate bone metabolism.

Effects of 1,25-dihydroxyvitamin D3 on the local bone renin-angiotensin system in a murine model of glucocorticoid-induced osteoporosis

“…mRNA expression levels of ACE, Ang II, AT1R, Ang type 2 receptor (AT2R), osteoprotegerin (OPG) and RANKL were measured using a qPCR kit [FastStart Universal SYBR-Green 1 PCR master mix (Rox); Roche Diagnostics, Indianapolis, IN, USA] and the gene-specific primers demonstrated in Table I. The qPCR reaction mixture consisted of a total reaction volume of 20 µl that contained 2.9 µl H 2 O, 1.0 µl cDNA sample, 5.0 µl 10X buffer, 7.0 µl 25 mM MgCl 2 , 1.0 µl 10 mM dNTPs, 0.8 µl forward primer (20 pmol/ml), 0.8 ml reverse primer (20 pmol/ml), 1.0 µl SYBR-Green and 0.5 µl Taq (5 U/ml) (22). A real-time PCR system (ABI7300; Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to run the following cycling conditions: 95°C for 10 min; followed by 40 cycles at 95–60°C for 15 sec, and the final extension at 60°C for 5 min.…”

“…A real-time PCR system (ABI7300; Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to run the following cycling conditions: 95°C for 10 min; followed by 40 cycles at 95–60°C for 15 sec, and the final extension at 60°C for 5 min. Relative expression levels were normalized to the internal control β-actin according to the 2 −∆∆Cq method and three repeats were performed (22–25). …”

“…Subsequently, slides were incubated with primary anti-Ang II (1:50; sc-20718; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and anti-ACE (ab11734; 1:100), anti-AT1R (ab92486; 1:1,000) and anti-AT2R antibodies (ab108252; 1:1,000) (all from Abcam, Cambridge, UK) overnight at 4°C. Following this, slides were incubated with secondary goat antibody (ab191866; 1:1,000; Abcam) and horseradish peroxidase-labeled streptavidin (Wuhan Boster Biological Technology, Ltd., Wuhan, China; catalog no., BA1088) for 1 h at 4°C (22). Subsequently, antibody binding was visualized using DAB-H 2 O 2 solution.…”

“…Stained tissues were mounted, following hematoxylin staining, and viewed under a light microscope (magnification, ×400; Olympus Corp., Tokyo, Japan). Cells were counted as previously described (22). The anti-ACE, -Ang II, -AT1R and -AT2R labeling indices were calculated using the following formula: ACE labeling index = number of ACE-positive cells/total number of cells per high power field × 100 (22).…”

“…Osteocytes were mononuclear cells filling the bone lacunae of the trabecular. All cells in the bone marrow were defined as bone marrow cells, regardless of cell type (22). …”