1. Volume 84, Issue 1, Pages 54-63 (July 2013)
Calcitriol restores renovascular function in estrogen-deficient rats through downregulation of cyclooxygenase-2 and the thromboxane-prostanoid receptor 
Jinghui Dong, Siu Ling Wong, Chi Wai Lau, Jian Liu, Yi-Xiang Wang, Zhen Dan He, Chi Fai Ng, Zhen Yu Chen, Xiaoqiang Yao, Aimin Xu, Xiaochen Ni, Hongyan Wang, Yu Huang 
Kidney International 
Volume 84, Issue 1, Pages (July 2013)
DOI: /ki
Copyright © 2013 International Society of Nephrology Terms and Conditions

2. Figure 1
Chronic calcitriol treatment improves endothelium-dependent relaxations during estrogen deficiency. (a, b) Renal arteries from ovariectomized (OVX) rats exhibited impaired acetylcholine (ACh)-induced relaxations, which were partially restored by oral treatment with calcitriol (n=8). (c) Endothelium-independent sodium nitroprusside (SNP)-induced relaxations were similar in all groups (n=5). Data are means±s.e.m. of 4–8 experiments. *P<0.05 vs. sham; #P<0.05 vs. OVX+vehicle. % Phe tone, percentage of tension with phenylephrine contraction.
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3. Figure 2
Magnetic resonance imaging on renal blood flow. (a) Representative images and (b, c) summarized data showing that calcitriol restored the renal blood flow in ovariectomized (OVX) rats. Data are means±s.e.m. of four experiments. *P<0.05 vs. sham-operated rats; #P<0.05 vs. OVX rats treated with vehicle.
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4. Figure 3
Inhibitors of cyclooxygenase-2 (COX-2) and thromboxane-prostanoid (TP) receptor improve endothelium-dependent relaxations. Impaired acetylcholine (ACh)-induced relaxations in ovariectomized (OVX) rat renal arteries were reversed acutely by (a) nonselective COX inhibitor indomethacin (3μmol/l), (b) COX-2 inhibitors celecoxib, DuP-697, and NS-398 (all at 3μmol/l), and (d) TP receptor antagonist S18886 (0.3μmol/l), but remained unaffected by (c) COX-1 inhibitor SC560 (10nmol/l). Data are means±s.e.m. of 3–6 experiments. *P<0.05 vs. control. % Phe tone, percentage of tension with phenylephrine contraction.
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5. Figure 4
Elevated expression level of cyclooxygenase-2 (COX-2) and thromboxane-prostanoid (TP) receptor is normalized by calcitriol treatment. The increased expression of (a) COX-2 and (b) TP receptor (TPR) in renal arteries from ovariectomized (OVX) rats was reduced by calcitriol, whereas (c) COX-1 expression was not changed in the three groups of rats. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Data are means±s.e.m. of 3–5 experiments. *P<0.05 vs. sham control; #P<0.05 vs. OVX+vehicle.
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6. Figure 5
Effects of ex vivo calcitriol treatment on renal arteries. (a, b) The acetylcholine (ACh)-induced relaxations were improved by a 12-h culture of ovariectomized (OVX) rat renal arteries with 100nmol/l calcitriol, but were (a, c) unaffected by acute 30-min exposure to calcitriol. Data are means±s.e.m. of 4–6 experiments. *P<0.05 vs. control. (d) The increased protein expression of thromboxane-prostanoid (TP) receptor (TPR) was reversed by a 12-h exposure to calcitriol or celecoxib. Data are means±s.e.m. of 3–4 experiments. *P<0.05 vs. sham; #P<0.05 vs. OVX. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; % Phe tone, percentage of tension with phenylephrine contraction.
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7. Figure 6
Thromboxane-prostanoid receptor (TPR) activation impairs vascular function in sham-operated rats. (a, b) Ex vivo 12-h incubation with 10nmol/l U46619 impaired acetylcholine (ACh)-induced relaxations and (c) elevated the expression of TPR in sham-operated rat renal arteries. These were reversed by cotreatment with calcitriol or S Data are means±s.e.m. of 4–6 experiments. *P<0.05 vs. control; #P<0.05 vs. U GAPDH, glyceraldehyde-3-phosphate dehydrogenase; % Phe tone, percentage of tension with phenylephrine contraction.
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8. Figure 7
Effect of calcitriol treatment on nitric oxide (NO) production in ovariectomized (OVX) rat aortic endothelial cells. (a) Confocal microscopy showed that acetylcholine (ACh)-stimulated NO production was reduced in primary aortic endothelial cells from OVX rats compared with sham-operated rats. Treatment (12h) with calcitriol, S18886, or celecoxib enhanced the NO production. Data are means±s.e.m. of five experiments. The elevated expression of (b) cyclooxygenase-2 (COX-2) and (c) thromboxane-prostanoid receptor (TPR) in these endothelial cells from OVX rats was normalized by a 12-h treatment with calcitriol, and of TPR by celecoxib as well (n=5). (d) COX-1 expression remained unchanged (n=3). *P<0.05 vs. sham; #P<0.05 vs. OVX. (e) Treatment (12h) with U46619 reduced ACh-stimulated NO production, whereas this effect was partially reversed by cotreatment with calcitriol in sham-operated rat aortic endothelial cells. Data are means±s.e.m. of 5 experiments. *P<0.05 vs. control; #P<0.05 vs. U DAF-FM DA, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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9. Figure 8
The effects of calcitriol are dose dependent. (a) Calcitriol reduced thromboxane-prostanoid receptor (TPR) expression level in U46619-treated renal arteries from sham-operated rats in a concentration-dependent manner, and (b) increased phosphorylated endothelial nitric oxide synthase (p-eNOS) level and (c, d) nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs) exposed to U (b, e) The effects of calcitriol were antagonized by a human-specific vitamin D receptor blocker TEI (f) U46619-inhibited NO production was sensitive to S18886 and celecoxib. Data are means±s.e.m. of four experiments. *P<0.05 vs. control; #P<0.05 vs. U46619-treated tissue or cells; §P<0.05 versus tissue or cells treated with U46619+calcitriol (100nmol/l). DAF-FM DA, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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10. Figure 9
Schematic diagram showing the alteration of renovascular reactivity under estrogen deficiency and the effects of calcitriol. Cyclooxygenase-2 (COX-2) expression is elevated under estrogen deficiency, resulting in the increased expression of thromboxane-prostanoid receptor (TPR). COX-2 products/TPR activation reduces nitric oxide (NO) bioavailability, leading to impaired endothelium-dependent relaxations. Calcitriol restores vascular function by normalizing the endothelial expression of COX-2 and TPR, thus preventing the TPR activation–induced NO reduction.
Kidney International  , 54-63DOI: ( /ki )
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