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 54-63 (July 2013) DOI: 10.1038/ki.2013.12 Copyright © 2013 International Society of Nephrology Terms and Conditions

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. Kidney International 2013 84, 54-63DOI: (10.1038/ki.2013.12) Copyright © 2013 International Society of Nephrology Terms and Conditions

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. Kidney International 2013 84, 54-63DOI: (10.1038/ki.2013.12) Copyright © 2013 International Society of Nephrology Terms and Conditions

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. Kidney International 2013 84, 54-63DOI: (10.1038/ki.2013.12) Copyright © 2013 International Society of Nephrology Terms and Conditions

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. Kidney International 2013 84, 54-63DOI: (10.1038/ki.2013.12) Copyright © 2013 International Society of Nephrology Terms and Conditions

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. Kidney International 2013 84, 54-63DOI: (10.1038/ki.2013.12) Copyright © 2013 International Society of Nephrology Terms and Conditions

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 S18886. Data are means±s.e.m. of 4–6 experiments. *P<0.05 vs. control; #P<0.05 vs. U46619. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; % Phe tone, percentage of tension with phenylephrine contraction. Kidney International 2013 84, 54-63DOI: (10.1038/ki.2013.12) Copyright © 2013 International Society of Nephrology Terms and Conditions

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. U46619. DAF-FM DA, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Kidney International 2013 84, 54-63DOI: (10.1038/ki.2013.12) Copyright © 2013 International Society of Nephrology Terms and Conditions

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 U46619. (b, e) The effects of calcitriol were antagonized by a human-specific vitamin D receptor blocker TEI-9647. (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. Kidney International 2013 84, 54-63DOI: (10.1038/ki.2013.12) Copyright © 2013 International Society of Nephrology Terms and Conditions

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 2013 84, 54-63DOI: (10.1038/ki.2013.12) Copyright © 2013 International Society of Nephrology Terms and Conditions