Journal of Steroid Biochemistry & Molecular Biology 148 (2015) 202–209
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Review
Different vitamin D receptor agonists exhibit differential effects on endothelial function and aortic gene expression in 5/6 nephrectomized rats J. Ruth Wu-Wong a, * , Xinmin Li b , Yung-wu Chen a a b
Vidasym, Chicago, IL, United States Department of Pathology & Laboratory Medicine, University of California, Los Angeles, CA, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 July 2014 Received in revised form 25 November 2014 Accepted 3 December 2014 Available online 10 December 2014
Endothelial dysfunction, common in chronic kidney disease (CKD), significantly increases cardiovascular disease risk in CKD patients. This study investigates whether different vitamin D receptor agonists exhibit different effects on endothelial function and on aortic gene expression in an animal CKD model. The 5/6 nephrectomized (NX) rat was treated with or without alfacalcidol (0.02, 0.04 and 0.08 mg/kg), paricalcitol (0.04 and 0.08 mg/kg), or VS-105 (0.004, 0.01 and 0.16 mg/kg). All three compounds at the test doses suppressed serum parathyroid hormone effectively. Alfacalcidol at 0.08 mg/kg raised serum calcium significantly. Endothelial function was assessed by pre-contracting thoracic aortic rings with phenylephrine, followed by treatment with acetylcholine or sodium nitroprusside. Uremia significantly affected endothelial-dependent aortic relaxation, which was improved by all three compounds in a dosedependent manner with alfacalcidol and paricalcitol exhibiting a lesser effect. DNA microarray analysis of aorta samples revealed that uremia impacted the expression of numerous aortic genes, many of which were normalized by the vitamin D analogs. Real-time RT-PCR analysis confirmed that selected genes such as Abra,Apoa4, Fabp2, Hsd17b2, and Hspa1b affected by uremia were normalized by the vitamin D analogs with alfacalcidol exhibiting less of an effect. These results demonstrate that different vitamin D analogs exhibit different effects on endothelial function and aortic gene expression in 5/6 NX rats. This article is part of a Special Issue entitled ‘17th Vitamin D Workshop’. ã 2014 Elsevier Ltd. All rights reserved.
Keywords: Alfacalcidol Aortic gene expression Endothelial function Kidney disease Paricalcitol Vitamin D analogs Vitamin D receptor VS-105
Contents 1. 2.
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Sub-total nephrectomized rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Measurements of physiological parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Vascular function studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Microarray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Real-time RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Effect of uremia and VDRM treatment on blood chemistry and endothelial function 3.2. Effects of uremia and VDRMs on aortic gene expression via DNA microarray analysis
Abbreviation: Ca, calcium; CKD, chronic kidney disease; NX, nephrectomized; PTH, parathyroid hormone; Pi, phosphorus/phosphate; VDR, vitamin D receptor; VDRM, VDR modulator. * Corresponding author at: 2201 W. Campbell Park Dr., Suite 13, Chicago, IL 60612, United States. Tel.: +1 847-680-6072. E-mail address: [email protected] (J. R. Wu-Wong). http://dx.doi.org/10.1016/j.jsbmb.2014.12.002 0960-0760/ ã 2014 Elsevier Ltd. All rights reserved.
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4.
3.3. Confirmation of gene microarray results by real-time RT-PCR Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Chronic kidney disease (CKD) is becoming a serious public health problem. Based on information provided by the National Kidney Foundation, 26 million people in America (13% of the US population) have CKD and the number is increasing rapidly due to the aging population and the epidemics of Type 2 diabetes and hypertension [1]. CKD patients experience a high mortality rate from cardiovascular diseases [2–4], and the mortality risk increases with kidney disease progression [4]. Endothelial dysfunction is usually present before the onset of clinical left ventricular hypertrophy, a risk factor for stroke, coronary heart disease, and cardiovascular disease in general [5]. Systemic endothelial dysfunction is frequently found in CKD [6,7], which contributes to the severity of cardiovascular complications in CKD patients who usually have hypertension [8] and/or diabetes [9], two of the conventional cardiovascular risk factors known to be associated with endothelial dysfunction. In addition, other renal specific mechanisms that disrupt the delicate balance among inflammation, fibrinolysis, oxidative stress, thrombosis and vasodilation maintained by the endothelium may contribute to endothelial dysfunction in CKD. During the course of CKD progression, a decrease in the serum 1,25-dihydroxyvitamin D3 levels is one of the first serum parameters to be altered [10]. It has been postulated that deficient vitamin D receptor (VDR) activation may be one of the renal specific risk factors for endothelial dysfunction in CKD [11]. Evidence suggests that VDR may play a role in modulating cardiovascular functions [12]. Vitamin D and vitamin D analogs that activate VDR (VDR agonists or modulators, VDRMs) have been shown to modulate inflammation, thrombosis, and vasodilation, all of which are important risk factors associated with endothelial dysfunction [13]. Previously we [14,15] and others [16] have reported that uremia significantly reduces acetylcholine-evoked relaxation in arteries prepared from the 5/6 NX uremic rat, indicating compromised endothelial function. We have also demonstrated that, while both VS-105 and paricalcitol improved endothelial function in 5/6 NX rats in a dose-dependent manner [14,15], the efficacious and hypercalcemic dose range was not clearly separated for paricalcitol. It is not known whether all vitamin D analogs exhibit similar effects on endothelial function. Therefore, this study compared the effects of alfacalcidol, paricalcitol, and VS-105 on endothelial function in 5/6 NX rats (see Supplementary Fig. S1 for the structures of the three compounds). Alfacalcidol (1-hydroxycholecalciferol) is a precursor of calcitriol (1,25(OH)2D3), and is converted into calcitriol in vivo. To investigate the mechanism of action, DNA microarray technology was employed to compare the effects of the three VDRMs on aortic gene expression. 2. Methods
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methylenecyclohexane-1,3-diol) were made by Vidasym. See Supplementary Fig. S1 for the structures of alfacalcidol, paricalcitol, and VS-105. Other reagents were of analytical grade. 2.2. Sub-total nephrectomized rats The nephrectomy was performed on male, Sprague Dawley rats weighing 200–220 g using a standard two-step surgical ablation procedure [17]. Rats were maintained on a normal diet containing 1% calcium and 0.7% phosphorus. At 6 weeks after the second surgery, when uremia was firmly established (as indicated by elevated serum creatinine and BUN), rats were treated with vehicle (5% ethanol + 95% propylene glycol, 0.4 mL/kg) or test agent (in vehicle), i.p. 3/week for 12 days. Each group consisted of 8–12 animals. Untreated, age-matched sham rats served as controls. Blood was drawn on Days 0 (24 h before the first dose) and 13 (24 h after the last dose), and assayed for creatinine, BUN, PTH, total calcium (Ca), and phosphorus/phosphate (Pi). At the end of the study, aorta was collected for further analyses. The animal studies were conducted under the auspice of the Office of Animal Care and Institutional Biosafety, University of Illinois at Chicago. The studies conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). 2.3. Measurements of physiological parameters Serum Ca was measured using the Stanbio LiquiColor calcium assay kit (Boerne, TX). Serum PTH was measured using a rat intact PTH ELISA kit obtained from Immutopics (San Clemente, CA). Serum creatinine, BUN and phosphorus/phosphate were measured using a standard chemistry analyzer. 2.4. Vascular function studies Thoracic aortas from rats were excised in a cold modified Krebs solution (see below) and a 3 mm aortic ring was suspended in 10 mL tissue baths under 0.5 g of resting tension in a modified Krebs solution containing (g/L): NaCl 6.9169, KCl 0.3499, NaHCO3 2.0998, MgSO4 0.2901, KH2 PO4 0.1604, CaCl2 0.2663, glucose 1.9994, EDTA 0.026, equilibrated with 5% CO2–95% O2 (pH 7.4 at 37 C). Aortas were sensitized by the addition of phenylephrine (PE, 3 mM) with 10 min washouts between intervals. Aortas were pre-contracted with PE (3 mM), and the endothelium-dependent vasodilator acetylcholine (ACh) was added in half-log or log increments (10 9 –10 4 mol/L) at 3–5 min intervals, allowing time for the effect of ACh to plateau. After a 60 min washout, aortas were pre-contracted with PE (3 mM) and subsequently treated with endothelium-independent vasodilator sodium nitroprusside (SNP; 10 9–10 4 mol/L) at 3–5 min intervals, allowing time for the effect to plateau. Data were recorded with the BL-420F data acquisition and analysis system.
2.1. Materials Paricalcitol (19-nor-1a,25(OH)2D2), alfacalcidol (1-hydroxycholecalciferol), and VS-105 ((1R,3R)-5-((E)-2-((3aS,7aS)-1-((R)-1((S)-3-hydroxy-2,3-dimethylbutoxy)ethyl)-7a-methyldihydro1H-inden 4(2H,5H,6H,7H,7aH)-ylidene) ethylidene)-2-
2.5. Microarray To obtain enough RNA, two aortic RNA samples in the same treatment group (with the same amount of RNA from each sample) were randomly combined and three pooled samples per treatment
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group were randomly selected for the microarray analysis. Thus, representative samples at n = 3 were achieved for each of the 5 treatment groups: sham, NX-vehicle, NX-paricalcitol at 0.08 mg/kg, NX-alfacalcidol at 0.04 mg/kg, and NX-VS-105 at 0.01 mg/kg. The dose for each compound for the microarray analysis was chosen based on its efficacy in PTH suppression without a significant elevation in serum calcium. The RNAs were intact as judged by Agilent 2100 DNA analyzer. Total RNA (200 ng per sample) from each sample was used to prepare Cy3-labeled cRNA target using standard Agilent protocols. Prepared cRNA targets were of good quality and quantity. The Agilent rat 8 60 k gene expression array was used and 600 ng cRNA target was applied to each array. After hybridization and chip scanning, the quality control data report (i.e., noise, background) demonstrated that every array passed all quality criteria. Raw data were loaded into Partek genomics Suite 6.0. Thresholds for selecting significant genes were set at 1.5-fold or 1.5-fold and p 0.01. Genes that met both criteria simultaneously were considered as exhibiting significant changes. Hierarchical clustering and gene ontology analysis were used to assess the function of the regulated genes. The raw data have been submitted to a public repository (Gene Expression Omnibus (GEO) at http://www.ncbi.nlm.nih.gov/geo/). 2.6. Real-time RT-PCR Real-time reverse transcription (RT)-PCR was performed using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). Each sample has a final volume of 25 mL containing 200 ng of mRNA, 100 nM (final concentration) each of the forward and reverse PCR primers and 250 nM (final concentration) of the TaqManTM probe (Applied Biosystems). Temperature conditions consisted of a step of 30 min at 48 C and a step of 10 min at 95 C, followed by 45 cycles of 60 C for 1 min and 95 C for 15 s. Data were collected during each extension phase of the PCR reaction and analyzed using a software package (Applied Biosystems). Threshold cycles were determined for each gene. 2.7. Data analysis For serum chemistry data, group mean SEM are presented. Differences across different treatment groups were assessed using a one-way ANOVA followed by a Dunnett’s post-hoc test. A t-test was used to assess differences between two treatment groups. For vascular function, ACh and SNP-induced relaxation were calculated as the % relaxation of the PE-induced pre-contraction. Differences
in vascular function were determined using a two-way ANOVA, followed by a Bonferroni post-hoc test. 3. Results 3.1. Effect of uremia and VDRM treatment on blood chemistry and endothelial function As shown in Table 1, the serum creatinine and BUN levels were significantly elevated in 5/6 nephrectomized (NX) rats compared to sham rats on both Day 0 (6 weeks after surgery) and Day 13 (8 weeks after surgery), indicating established uremia. The three VDRMs had no significant effect on either BUN or creatinine (vs. Day 0) except that VS-105 at 0.16 mg/kg significantly reduced serum BUN levels. Serum phosphorus/phosphate (Pi) and calcium (Ca) levels were not significantly different in 5/6 NX rats vs. sham, and the compounds had no significant effect on the Pi and Ca levels except that alfacalcidol at 0.08 mg/kg significantly elevated serum Ca levels, and paricalcitol at 0.08 mg/kg exhibited a modest hypercalcemic effect. Serum PTH levels were significantly elevated in 5/6 NX rats treated with vehicle, and were effectively suppressed by the three compounds at all tested doses. Fig. 1A shows that the maximal aortic relaxation response to acetylcholine was significantly reduced in the aorta from 5/6 NX vehicle-treated rats ( 62.1 4.6% in NX vs. 87.0 1.3% in sham rats), indicating compromised endothelial function. Fig. 1B shows that the aortic relaxation produced by sodium nitroprusside (SNP, an endothelium-independent vasodilator) was similar between the sham and 5/6 NX rats, indicating that the smooth muscle relaxation response is functional. Fig. 1A demonstrates that paricalcitol after two weeks of treatment produced a modest, yet dose-dependent improvement in acetylcholine-induced (endothelium-dependent) aortic relaxation. The maximal relaxation response to acetylcholine with 0.04 and 0.08 mg/kg of paricalcitol was at 68.7 2.2% and 71.0 2.8%, respectively. Consistent with our previous report [14], paricalcitol had no effect on SNP-induced relaxation (Fig. 1B) As a comparison, Fig. 2A shows that 0.04 mg/kg of alfacalcidol after 2-weeks of treatment exhibited a modest effect on the maximal relaxation response to acetylcholine ( 69.8 2.4%), while 0.02 mg/kg of alfacalcidol showed minimal effects. A higher dose (0.08 mg/kg) that induced hypercalcemia was then tested, which further improved the maximal relaxation response to acetylcholine at 76.8 14.1% (vs. the maximal aortic relaxation response to acetylcholine in sham rats at 87.0 1.3%, and in
Table 1 Effects of VDRMs on physiological parameters in sham vs. 5/6 nephrectomized rats. Parameters
Sham 5/6 NX-Vehicle Pari, 0.04 mg/kg Pari, 0.08 mg/kg Alfa, 0.02 mg/kg Alfa, 0.04 mg/kg Alfa, 0.08 mg/kg VS-105, 0.004 mg/kg VS-105 0.01 mg/kg VS-105 0.16 mg/kg
Creatinine (mg/dL)
BUN (mg/dL)
Serum Pi (mg/dL)
Serum Ca (mg/dL)
Serum PTH (pg/mL)
Day 0
Day 13
Day 0
Day 13
Day 0
Day 13
Day 0
Day 13
Day 0
Day 13
0.32 0.02 0.93 0.04a 0.76 0.05a 0.76 0.04a 0.81 0.02a 0.78 0.03a 0.76 0.06a 0.90 0.02a
0.33 0.01 1.05 0.08a 0.86 0.05a 0.90 0.04a,b 0.88 0.03a 0.87 0.04a 0.89 0.11a 0.93 0.03a
15.7 0.5 41.1 1.7a 38.2 2.7a 42.4 2.2a 44.0 2.0a 40.8 1.9a 44.1 3.2a 34.2 1.3a
15.2 0.5 43.9 2.5a 39.8 2.9a 42.0 2.3a 39.8 2.4a 38.8 2.1a 43.7 7.8a 33.0 1.6a
7.5 0.3 7.9 0.3 7.0 0.5 7.2 0.2 8.2 0.2 7.6 0.2 8.2 0.1 7.7 0.8
8.1 0.4 8.1 0.6 6.7 0.8 7.9 0.3 8.4 0.9 7.7 0.3 8.5 0.2 8.3 0.1
9.8 0.2 10.3 0.2 10.3 0.4 9.8 0.3 9.9 0.3 10.2 0.2 10.7 0.1 10.4 0.1
9.9 0.4 10.4 0.7 10.5 0.4 10.9 0.3b 9.3 0.3 10.7 0.2 12.3 0.7b 10.3 0.1
109 8 415 38 379 8 455 43 388 18 460 46 516 66 324 28
126 11 482 40 92 6d 117 41d 98 10d 163 42d 54 30d 216 23b
0.82 0.04a
0.82 0.04a
34.0 1.5a
30.8 1.7a
7.5 0.2
7.3 0.2
9.9 0.1
10.0 0.2
361 22
137 9d
0.92 0.03a
0.93 0.02a
37.8 1.4a
32.3 1.2a,c
7.5 0.2
7.1 0.1
10.6 0.1
10.0 0.1
386 24
96 8d
Rats were treated as in Section 2.1. Data presented are mean SEM (n = 8–12 per group). Pari: paricalcitol; Alfa: alfacalcidol. a p < 0.001 vs. sham Day 0. b p < 0.05 vs. same group Day 0. c p < 0.01 vs. same group Day 0. d p < 0.001 vs. same group Day 0.
J.R. Wu-Wong et al. / Journal of Steroid Biochemistry & Molecular Biology 148 (2015) 202–209
[(Fig._1)TD$IG]
(B)
0 ***
% Relaxation
-25
***
***
*** *** * ***
-50
-75
-100 -10
Sham 5/6 NX Vehicle Pari-0.04 Pari-0.08
-9
*** *** *** ### *** *** *** *** *** *** ***
-8 -7 -6 Ach [log, M]
-5
0
-25
% Relaxation
(A)
-50
-75
-100 -10
-4
205
Sham 5/6 NX Vehicle Pari-0.04 Pari-0.08
-9
-8
-7
-6
-5
-4
SNP [log, M]
Fig. 1. Effect of paricalcitol on aortic relaxation in 5/6 nephrectomized rats. Sham and 5/6 NX rats were treated with vehicle or paricalcitol at indicated doses as described in Section 2.1. Aortic rings were pre-contracted with phenylephrine (PE, 3 mM), and the endothelium-dependent vasodilator acetylcholine (Ach) was added (doses as shown) at 3–5 min intervals. Afterwards, aortic rings were pre-contracted with PE (3 mM) and subsequently treated with the endothelium-independent vasodilator sodium nitroprusside (SNP, doses as shown) at 3–5 min intervals. (A) Acetylcholine-evoked relaxation. (B) SNP-evoked relaxation. ###p < 0.001 vs. sham (at all Ach doses); *p < 0.05, **p < 0.01, ***p < 0.001 vs. NX-vehicle at the indicated Ach dose.
[(Fig._2)TD$IG] 0
(B)
-25 *** -50 -75 -100 -10
Sham 5/6 NX Vehicle Alfa-0.02 Alfa-0.04 Alfa-0.08
-9
***
*** *** ***
-8 -7 -6 Ach [log, M]
###
*** *** *** *** ***
-5
-4
% Relaxation
% Relaxation
(A)
0
### #
*
-25
###
*** -50 -75 -100 0 -10
Sham 5/6 NX Vehicle VS-105 0.004 g/kg VS-105 0.01 g/kg VS-105 0.16 g/kg
-9
-8
###
* ***
-7 -6 Ach [log, M]
### ###
* ***
*** ***
-5
-4
Fig. 2. Effect of alfacalcidol and VS-105 on aortic relaxation in 5/6 nephrectomized rats. Sham and 5/6 NX rats were treated with vehicle or test agents at indicated doses as described in Section 2.1. Aortic rings were pre-contracted with phenylephrine (PE, 3 mM), and the endothelium-dependent vasodilator acetylcholine was added as in Fig. 1. Alfacalcidol and VS-105 were tested in two independent studies using two different groups of 5/6 NX rats. (A) Alfacalcidol. ###p < 0.001 at all Ach doses; ***p < 0.001 vs. NXvehicle at the indicated Ach dose. (B) VS-105. #p < 0.05, ###p < 0.001 vs. sham at the indicated Ach dose; *p < 0.05, ***p < 0.001 vs. NX-vehicle at the indicated Ach dose.
vehicle-treated rats at 62.1 4.6%). Alfacalcidol had no significant effects on SNP-induced relaxation (data not shown). Consistent with our previous report [15], Fig. 2B demonstrates that, VS-105 (i. p., 3/week, for 2 weeks) exerted a dose-dependent improvement in acetylcholine-induced aortic relaxation. The 0.16 mg/kg dose normalized acetylcholine-induced aortic relaxation, while the low dose at 0.004 mg/kg did not exhibit a significant effect. The maximal relaxation response to acetylcholine was at 54.3 8.2% and 78.2 12.9% for 0.01 and 0.16 mg/kg of VS-105, respectively (vs. the maximal aortic relaxation response to acetylcholine at 78.5 5.8% in sham rats, and at 34.5 7.4% in vehicle-treated rats). VS-105 also had no significant effect on SNP-induced endothelial-independent relaxation (data not shown).
3.2. Effects of uremia and VDRMs on aortic gene expression via DNA microarray analysis The hierarchical clustering of genes (with the expression value of each gene in each sample) suggests that uremia exerts a significant effect on aortic gene expression, and all three compounds at the tested dose were able to normalize a subset of genes affected by uremia (see Supplementary Fig. S2). Using thresholds for selecting significant genes at 1.5-fold or 1.5-fold of change and p 0.01, there were 234 genes found to exhibit significant changes in 5/6 NX uremic rats (vs. sham). When the selection criteria were further narrowed down to be at 2-fold or 2-fold of change and p 0.01, 89 genes were tabulated to
Table 2 Aortic genes differentially regulated by uremia and three VDRMs. NX onlya (vs. sham)
NX + paricalcitol (vs. sham)
NX + alfacalcidol (vs. sham)
NX + VS-105 (vs. sham)
89 genes affected (p 0.01, 2-fold) Up-regulated: 68 genes. Down-regulated: 21 genes. –
56 genes affected (p 0.01, 2-fold). Up-regulated: 42 genes. Down-regulated: 14 genes. 33 genes normalized (vs. NX).
46 genes affected (p 0.01, 2-fold). Up-regulated: 35 genes. Down-regulated: 11 genes. 43 genes normalized (vs. NX).
46 genes affected (p 0.01, 2-fold). Up-regulated: 28 genes. Down-regulated: 18 genes. 43 genes normalized (vs. NX)
a
NX only: 5/6 nephrectomized rats treated with vehicle.
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Table 3 Gene ontology analysis of aortic genes affected by uremia and VDRM treatment. Category
Term
Count
p-Value
Benjamini
Normalized by VDRMs
KEGG_PATHWAY INTERPRO KEGG_PATHWAY KEGG_PATHWAY GOTERM_BP_FAT SP_PIR_KEYWORDS GOTERM_BP_FAT KEGG_PATHWAY INTERPRO INTERPRO GOTERM_CC_FAT GOTERM_CC_FAT GOTERM_BP_FAT KEGG_PATHWAY INTERPRO INTERPRO KEGG_PATHWAY
Type I diabetes mellitus MHC class I, alpha chain, C-terminal Graft vs. host disease Allograft rejection Immune response MHC I Antigen processing and presentation of peptide antigen via MHC class I Antigen processing and presentation MHC class I, alpha chain, alpha1 and alpha2 MHC class I-like antigen recognition MHC class I protein complex MHC protein complex Antigen processing and presentation of peptide antigen Autoimmune thyroid disease Immunoglobulin C1-set Immunoglobulin/major histocompatibility complex, conserved site Cell adhesion molecules (CAMs)
10 7 9 9 16 7 7 10 8 8 8 8 7 8 8 8 10
3.2E-11 1.2E-10 3.2E-10 4.4E-10 8.1E-10 8.9E-10 9.8E-10 1.1E-9 1.4E-9 3.1E-9 4.8E-9 2.1E-8 3.0E-8 4.4E-8 1.1E-7 1.1E-7 1.1E-7
1.8E-9 1.9E-8 8.7E-9 8.0E-9 8.9E-7 1.4E-7 5.4E-7 1.5E-8 1.1E-7 1.6E-7 7.7E-7 1.7E-6 1.1E-5 4.9E-7 4.4E-6 4.4E-6 1.0E-6
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
have significant changes in 5/6 NX uremic rats (vs. sham) as summarized in Table 2. Out of these 89 genes, 68 genes were up-regulated and 21 genes were down-regulated. When the data set was analyzed using DAVID (http://david.abcc.ncifcrf.gov/) to assess the general effects of uremia, a number of functionally related clusters were revealed. The top 18 gene ontology categories are shown in Table 3. Among the 89 genes affected by uremia, paricalcitol at 0.08 mg/kg normalized 33 genes, alfacalcidol at 0.04 mg/kg normalized 43 genes, and VS-105 at 0.01 mg/kg also normalized 43 genes (Table 2). There were 22 genes normalized by all three compounds (based on p value) as listed in Table 4. Four genes were normalized by alfacalcidol alone and four genes were normalized by VS-105 alone (based on p value) as listed in Table 5. When the gene data set normalized by all three VDRMs was analyzed using DAVID (http://david.abcc.ncifcrf.gov/), the functionally related clusters were similar to those shown in Table 3, suggesting that VDRM treatment was able to negate the uremic effect.
Table 4 Aortic genes that are normalized by all three VDRMs. Systematic name
ID
NX
NM_001164656 NM_021693 ENSRNOT00000001579 NM_019328 ENSRNOT00000036995 NM_001031642 NM_021836 ENSRNOT00000024094 NM_212504 NM_138525 NM_138541 NM_019287 NM_013183 NM_147206 NM_024391 NM_053977 NM_012975 NM_001107015 NM_013068 TC595358 NM_012738 NM_012737 NM_031669
Fcgbpl1 2.29 Sik1 2.36 Nr4a2 2.65 Adamtsl2 2.69 Serpinb1a 2.69 Junb 3.23 Sct 4.48 Hspa1b 5.26 Mucdhl 5.88 Epcam 6.01 Apob 6.61 Mep1b 7.34 Cyp3a9 8.41 Hsd17b2 9.46 Cdh17 10.48 Lgals4 10.72 Tm4sf5 11.14 Fabp2 11.43 TC595358 13.78 Apoa1 14.97 Apoa4 18.82 Prap1 29.55
Alfa 1.06 1.10 1.55 1.27 1.11 2.69 1.98 3.04 1.34 1.22 1.88 1.45 1.57 1.37 1.46 1.22 1.87 2.58 1.27 2.50 3.31 4.13
Pari 1.01 1.12 1.60 1.78 1.74 2.76 1.99 1.51 1.21 1.53 1.00 1.91 2.22 2.02 1.76 2.65 2.05 1.10 1.33 1.08 1.24 1.28
VS-105 1.20 1.18 1.02 1.64 1.88 1.65 1.87 1.28 1.27 3.04 1.17 2.89 3.08 2.73 2.34 2.38 1.30 1.69 1.37 1.56 1.86 1.82
Note: “Normalization” was based on p value changing from 0.01. NX: 5/6 nephrectomized rats treated with vehicle. Alfa: alfacalcidol; Pari: paricalcitol.
3.3. Confirmation of gene microarray results by real-time RT-PCR As a validation of the results, we employed real-time RT-PCR to examine the expression of 5 selected genes (Abra, NM_175844, actin-binding Rho activating protein; Apoa4, NM_012737, apolipoprotein A-IV; Fabp2, NM_013068, fatty acid binding protein 2; Hsd17b2, NM_024391; hydroxysteroid (17-beta) dehydrogenase 2; Hspa1b, NM_212504, heat shock 70 kDa protein 1B). Fig. 3A shows that the expression level of Abra was elevated in 5/6 NX rats, which was further increased in the paricalcitol and alfacalcidol groups, but not significantly changed in the VS-105 group. Consistent with the microarray analysis, Fig. 3B shows that Apoa4 was elevated in 5/6 NX rats; paricalcitol and VS-105 effectively reduced Apoa4 to the sham level, while alfacalcidol had lesser effects. The Q-PCR results confirmed that Fabp2 was elevated in 5/6 NX rats; all three compounds significantly reduced Fabp2, but alfacalcidol had lesser effects (Fig. 3C). Fig. 3D shows that the expression level of Hsd17b2 was low, but the results are consistent with the microarray analysis that Hsd17b2 was elevated in 5/6 NX rats. Paricalcitol and VS-105 reduced Hsd17b2 to the sham level, while alfacalcidol had lesser effects. Fig. 3E shows that all three compounds at the tested doses reduced the expression of Hspa1b with the most effect observed in the VS-105 group. 4. Discussion We have demonstrated in this report that alfacalcidol, paricalcitol and VS-105 all improve endothelial function in 5/6 NX rats in a dose dependent manner independent of their effects on serum calcium and PTH, but alfacalcidol at nonTable 5 Aortic genes that are normalized by alfacalcidol alone (top 4 genes) or VS-105 alone (bottom five genes). Systematic name
ID
NX
Alfa
Pari
VS-105
NM_134371 NM_133298 NM_001031652 XM_342027
Trpm8 Gpnmb St6galnac2 Ms4a4a
5.07 2.27 2.39 2.83
2.22 1.81 1.39 1.60
5.69 2.57 2.11 2.70
6.44 2.74 3.14 2.97
NM_012797 NM_012620 NM_001108621 NM_175844 ENSRNOT00000001953
Id1 Serpine1 Fam180a Abra Srcrb4d
2.53 4.47 2.11 5.94 5.07
2.70 4.05 2.17 5.83 3.87
2.13 3.82 2.30 4.66 2.19
2.00 2.80 1.87 2.30 1.19
Note: “Normalization” was based on p value changing from 0.01. NX: 5/6 nephrectomized rats treated with vehicle. Alfa: alfacalcidol; Pari: paricalcitol.
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hypercalcemic doses exhibits less of an effect on endothelial function when compared with paricalcitol and VS-105. The current study shows that PTH suppression is not necessarily linked to endothelial functional improvement in uremic rats since all three VDRMs at the tested doses suppress PTH effectively and to similar levels, but they exhibited different effects on endothelial function. Previously we have also shown that cinacalcet, a calcimimetic, suppresses PTH effectively, but does not improve endothelial function [14]. These observations seem consistent with clinical observations in the past few years that the survival and cardiovascular benefit of vitamin D analog therapy in CKD patients is observed across different serum PTH levels and different VDRMs may exhibit differential effects [18–33]. However, it is worth noting that others have shown that elevated PTH may play a role in various cardiovascular disorders including abnormal vasodilation [34]. Previously Nilsson et al. have also reported that, in patients with primary hyperparathyroidism, parathyroidectomy significantly improved endothelial vasodilatory function [35]. Data from the current study suggest that changes in serum calcium do not seem to affect endothelial function, which contrasts with the observation made by Jolma et al. [36] that a high calcium diet, leading to increased serum calcium, enhanced the resistance to artery relaxation in 5/6 NX rats. In this study alfacalcidol at 0.08 mg/kg raised serum calcium significantly, yet its effect on improving endothelial function was greater than the effect at lower doses that did not affect serum calcium, suggesting that the higher the VDRM dose, the greater is its effect on endothelial function. At the same time, it is important to consider that hypercalcemia interferes with numerous physiological functions such as bone formation, hormone release, muscle contraction, and nerve and
207
brain functions. Moreover, extreme and/or persistent hypercalcemia can be lethal. Therefore, any degree of hypercalcemia is a condition to be avoided. In clinical setting, serum calcium is frequently monitored and VDRM doses are also frequently titrated in order to achieve PTH suppression without raising serum calcium. Our results suggest that a VDRM with a wider therapeutic index that can be administered at high doses without causing hypercalcemia may exhibit a greater degree of beneficial cardiovascular effects. In our previous study testing the effect of VDRMs on modulating gene expression in human coronary artery smooth muscle cells [37,38], we observed that a large cluster of genes involved in cell differentiation/proliferation were modulated when proliferating smooth muscle cells were treated by VDRMs. In addition, several cardiovascular-related genes were also affected by VDRMs. In this current study, we observed that uremia exerts a detrimental impact on aortic gene expression in 5/6 NX rats. Gene ontology analysis reveals that many of the altered genes are linked to the immune system, which is mostly corrected by VDRM treatment. When examined in more detail, several genes linked to oxidative stress, inflammation, metabolic syndrome, and hormone functions including Apoa4, FABP2, Hsd17b2, Abra, and Hspa1b were identified. Previously Apoa4 (NM_012737; apolipoprotein A-IV) was known to be involved in triglyceride-rich lipoprotein metabolism and reverse cholesterol transport [39]. More recently, it has been suggested that Apoa4 may modulate oxidative stress [40] and play a role in coronary artery calcification [41]. FABP2 (NM_013068; fatty acid binding protein 2), known to regulate the intracellular transport of long-chain fatty acids and their acyl-CoA esters, belongs to the family of fatty acid binding proteins, of which
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Fig. 3. Effects of VDRMs on mRNA expression of selected genes. RNA was isolated from aorta in sham, 5/6 NX rats treated with vehicle, and 5/6 NX rats treated with paricalcitol (0.08 mg/kg), alfacalcidol (0.04 mg/kg), or VS-105 (0.01 mg/kg). Each group consisted of 8–12 aortic RNA samples (one sample per rat). The mRNA levels of target genes were analyzed by real-time RT-PCR. GADPH was used for normalization. (A) Abra. (B) Apoa4. (C) Fabp2. (D) Hsd17b2. (E) Hspa1b. Statistical analysis was performed by unpaired t-test. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. sham; *p < 0.05, **p < 0.01, ***p < 0.001 vs. NX-vehicle. Pari: paricalcitol. Alfa: alfacalcidol.
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[(Fig._4)TD$IG]
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. jsbmb.2014.12.002. References
Fig. 4. Possible mechanisms for the effects of uremia and VDRM treatment in aortic endothelial function. Uremia is associated with inflammation, oxidative stress, abnormal metabolic pathways, and de-regulated hormone functions, leading to endothelial dysfunction, which is largely reversed by VDRM treatment.
several are associated with the metabolic syndrome [42]. FABP2 A54T polymorphism has been linked to impaired glucose tolerance, obesity, altered plasma lipids and lipoproteins [43]. Hsd17b2 (NM_024391; hydroxysteroid (17-beta) dehydrogenase 2), a member in the family of metabolic and regulatory enzymes [44], is known to regulate the biological activity of steroid hormones, including estrogen and androgens [45]. Abra (NM_175844; actin-binding Rho activating protein) may act as an activator of serum response factor (SRF)-dependent transcription through a mechanism involving RhoA and actin polymerization [46]. Targeted deletion of Abra in CL57BL/6 mice led to impaired arteriogenesis [47]. Last but not least, Hspa1b (NM_212504; heat shock 70 kDa protein 1B), a member of the heat shock protein 70 family, may be involved in the ubiquitinproteasome pathway through interaction with the AU-rich element RNA-binding protein 1 [48]. It has been shown that Hspa1b polymorphism is linked to familial forms of inflammatory dilated cardiomyopathy [49]. Taken together, data from the current study suggest that uremia significantly impacts several different mechanisms involved in the cardiovascular system including oxidative stress, inflammation, metabolic pathways and hormone functions, leading to de-regulation of numerous genes, which is largely reversed by VDRM treatment (Fig. 4). Interestingly, even among the five selected genes, data from real-time Q-PCR show that alfacalcidol at 0.04 mg/kg is less effective than VS-105 at 0.01 mg/kg although both compounds at the tested doses suppress serum PTH in a similar manner. In summary, VDR activation by alfacalcidol, paricalcitol and VS-105 significantly improve endothelial function in 5/6 NX uremic rat in a dose-dependent manner. More importantly, the effect of VDRMs on endothelial function is independent of PTH suppression. The results suggest that for VDRM therapy, optimal cardiovascular benefit may be best achieved at higher doses using a less hypercalcemic VDRM. Conflict of interest Chen and Wu-Wong work for Vidasym. Acknowledgements We are in debt to Dr. Jerry L. Wessale for his comments and suggestions.
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Review
Different vitamin D receptor agonists exhibit differential effects on endothelial function and aortic gene expression in 5/6 nephrectomized rats J. Ruth Wu-Wong a, * , Xinmin Li b , Yung-wu Chen a a b
Vidasym, Chicago, IL, United States Department of Pathology & Laboratory Medicine, University of California, Los Angeles, CA, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 July 2014 Received in revised form 25 November 2014 Accepted 3 December 2014 Available online 10 December 2014
Endothelial dysfunction, common in chronic kidney disease (CKD), significantly increases cardiovascular disease risk in CKD patients. This study investigates whether different vitamin D receptor agonists exhibit different effects on endothelial function and on aortic gene expression in an animal CKD model. The 5/6 nephrectomized (NX) rat was treated with or without alfacalcidol (0.02, 0.04 and 0.08 mg/kg), paricalcitol (0.04 and 0.08 mg/kg), or VS-105 (0.004, 0.01 and 0.16 mg/kg). All three compounds at the test doses suppressed serum parathyroid hormone effectively. Alfacalcidol at 0.08 mg/kg raised serum calcium significantly. Endothelial function was assessed by pre-contracting thoracic aortic rings with phenylephrine, followed by treatment with acetylcholine or sodium nitroprusside. Uremia significantly affected endothelial-dependent aortic relaxation, which was improved by all three compounds in a dosedependent manner with alfacalcidol and paricalcitol exhibiting a lesser effect. DNA microarray analysis of aorta samples revealed that uremia impacted the expression of numerous aortic genes, many of which were normalized by the vitamin D analogs. Real-time RT-PCR analysis confirmed that selected genes such as Abra,Apoa4, Fabp2, Hsd17b2, and Hspa1b affected by uremia were normalized by the vitamin D analogs with alfacalcidol exhibiting less of an effect. These results demonstrate that different vitamin D analogs exhibit different effects on endothelial function and aortic gene expression in 5/6 NX rats. This article is part of a Special Issue entitled ‘17th Vitamin D Workshop’. ã 2014 Elsevier Ltd. All rights reserved.
Keywords: Alfacalcidol Aortic gene expression Endothelial function Kidney disease Paricalcitol Vitamin D analogs Vitamin D receptor VS-105
Contents 1. 2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Sub-total nephrectomized rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Measurements of physiological parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Vascular function studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Microarray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Real-time RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Effect of uremia and VDRM treatment on blood chemistry and endothelial function 3.2. Effects of uremia and VDRMs on aortic gene expression via DNA microarray analysis
Abbreviation: Ca, calcium; CKD, chronic kidney disease; NX, nephrectomized; PTH, parathyroid hormone; Pi, phosphorus/phosphate; VDR, vitamin D receptor; VDRM, VDR modulator. * Corresponding author at: 2201 W. Campbell Park Dr., Suite 13, Chicago, IL 60612, United States. Tel.: +1 847-680-6072. E-mail address: [email protected] (J. R. Wu-Wong). http://dx.doi.org/10.1016/j.jsbmb.2014.12.002 0960-0760/ ã 2014 Elsevier Ltd. All rights reserved.
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4.
3.3. Confirmation of gene microarray results by real-time RT-PCR Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Chronic kidney disease (CKD) is becoming a serious public health problem. Based on information provided by the National Kidney Foundation, 26 million people in America (13% of the US population) have CKD and the number is increasing rapidly due to the aging population and the epidemics of Type 2 diabetes and hypertension [1]. CKD patients experience a high mortality rate from cardiovascular diseases [2–4], and the mortality risk increases with kidney disease progression [4]. Endothelial dysfunction is usually present before the onset of clinical left ventricular hypertrophy, a risk factor for stroke, coronary heart disease, and cardiovascular disease in general [5]. Systemic endothelial dysfunction is frequently found in CKD [6,7], which contributes to the severity of cardiovascular complications in CKD patients who usually have hypertension [8] and/or diabetes [9], two of the conventional cardiovascular risk factors known to be associated with endothelial dysfunction. In addition, other renal specific mechanisms that disrupt the delicate balance among inflammation, fibrinolysis, oxidative stress, thrombosis and vasodilation maintained by the endothelium may contribute to endothelial dysfunction in CKD. During the course of CKD progression, a decrease in the serum 1,25-dihydroxyvitamin D3 levels is one of the first serum parameters to be altered [10]. It has been postulated that deficient vitamin D receptor (VDR) activation may be one of the renal specific risk factors for endothelial dysfunction in CKD [11]. Evidence suggests that VDR may play a role in modulating cardiovascular functions [12]. Vitamin D and vitamin D analogs that activate VDR (VDR agonists or modulators, VDRMs) have been shown to modulate inflammation, thrombosis, and vasodilation, all of which are important risk factors associated with endothelial dysfunction [13]. Previously we [14,15] and others [16] have reported that uremia significantly reduces acetylcholine-evoked relaxation in arteries prepared from the 5/6 NX uremic rat, indicating compromised endothelial function. We have also demonstrated that, while both VS-105 and paricalcitol improved endothelial function in 5/6 NX rats in a dose-dependent manner [14,15], the efficacious and hypercalcemic dose range was not clearly separated for paricalcitol. It is not known whether all vitamin D analogs exhibit similar effects on endothelial function. Therefore, this study compared the effects of alfacalcidol, paricalcitol, and VS-105 on endothelial function in 5/6 NX rats (see Supplementary Fig. S1 for the structures of the three compounds). Alfacalcidol (1-hydroxycholecalciferol) is a precursor of calcitriol (1,25(OH)2D3), and is converted into calcitriol in vivo. To investigate the mechanism of action, DNA microarray technology was employed to compare the effects of the three VDRMs on aortic gene expression. 2. Methods
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methylenecyclohexane-1,3-diol) were made by Vidasym. See Supplementary Fig. S1 for the structures of alfacalcidol, paricalcitol, and VS-105. Other reagents were of analytical grade. 2.2. Sub-total nephrectomized rats The nephrectomy was performed on male, Sprague Dawley rats weighing 200–220 g using a standard two-step surgical ablation procedure [17]. Rats were maintained on a normal diet containing 1% calcium and 0.7% phosphorus. At 6 weeks after the second surgery, when uremia was firmly established (as indicated by elevated serum creatinine and BUN), rats were treated with vehicle (5% ethanol + 95% propylene glycol, 0.4 mL/kg) or test agent (in vehicle), i.p. 3/week for 12 days. Each group consisted of 8–12 animals. Untreated, age-matched sham rats served as controls. Blood was drawn on Days 0 (24 h before the first dose) and 13 (24 h after the last dose), and assayed for creatinine, BUN, PTH, total calcium (Ca), and phosphorus/phosphate (Pi). At the end of the study, aorta was collected for further analyses. The animal studies were conducted under the auspice of the Office of Animal Care and Institutional Biosafety, University of Illinois at Chicago. The studies conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). 2.3. Measurements of physiological parameters Serum Ca was measured using the Stanbio LiquiColor calcium assay kit (Boerne, TX). Serum PTH was measured using a rat intact PTH ELISA kit obtained from Immutopics (San Clemente, CA). Serum creatinine, BUN and phosphorus/phosphate were measured using a standard chemistry analyzer. 2.4. Vascular function studies Thoracic aortas from rats were excised in a cold modified Krebs solution (see below) and a 3 mm aortic ring was suspended in 10 mL tissue baths under 0.5 g of resting tension in a modified Krebs solution containing (g/L): NaCl 6.9169, KCl 0.3499, NaHCO3 2.0998, MgSO4 0.2901, KH2 PO4 0.1604, CaCl2 0.2663, glucose 1.9994, EDTA 0.026, equilibrated with 5% CO2–95% O2 (pH 7.4 at 37 C). Aortas were sensitized by the addition of phenylephrine (PE, 3 mM) with 10 min washouts between intervals. Aortas were pre-contracted with PE (3 mM), and the endothelium-dependent vasodilator acetylcholine (ACh) was added in half-log or log increments (10 9 –10 4 mol/L) at 3–5 min intervals, allowing time for the effect of ACh to plateau. After a 60 min washout, aortas were pre-contracted with PE (3 mM) and subsequently treated with endothelium-independent vasodilator sodium nitroprusside (SNP; 10 9–10 4 mol/L) at 3–5 min intervals, allowing time for the effect to plateau. Data were recorded with the BL-420F data acquisition and analysis system.
2.1. Materials Paricalcitol (19-nor-1a,25(OH)2D2), alfacalcidol (1-hydroxycholecalciferol), and VS-105 ((1R,3R)-5-((E)-2-((3aS,7aS)-1-((R)-1((S)-3-hydroxy-2,3-dimethylbutoxy)ethyl)-7a-methyldihydro1H-inden 4(2H,5H,6H,7H,7aH)-ylidene) ethylidene)-2-
2.5. Microarray To obtain enough RNA, two aortic RNA samples in the same treatment group (with the same amount of RNA from each sample) were randomly combined and three pooled samples per treatment
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group were randomly selected for the microarray analysis. Thus, representative samples at n = 3 were achieved for each of the 5 treatment groups: sham, NX-vehicle, NX-paricalcitol at 0.08 mg/kg, NX-alfacalcidol at 0.04 mg/kg, and NX-VS-105 at 0.01 mg/kg. The dose for each compound for the microarray analysis was chosen based on its efficacy in PTH suppression without a significant elevation in serum calcium. The RNAs were intact as judged by Agilent 2100 DNA analyzer. Total RNA (200 ng per sample) from each sample was used to prepare Cy3-labeled cRNA target using standard Agilent protocols. Prepared cRNA targets were of good quality and quantity. The Agilent rat 8 60 k gene expression array was used and 600 ng cRNA target was applied to each array. After hybridization and chip scanning, the quality control data report (i.e., noise, background) demonstrated that every array passed all quality criteria. Raw data were loaded into Partek genomics Suite 6.0. Thresholds for selecting significant genes were set at 1.5-fold or 1.5-fold and p 0.01. Genes that met both criteria simultaneously were considered as exhibiting significant changes. Hierarchical clustering and gene ontology analysis were used to assess the function of the regulated genes. The raw data have been submitted to a public repository (Gene Expression Omnibus (GEO) at http://www.ncbi.nlm.nih.gov/geo/). 2.6. Real-time RT-PCR Real-time reverse transcription (RT)-PCR was performed using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). Each sample has a final volume of 25 mL containing 200 ng of mRNA, 100 nM (final concentration) each of the forward and reverse PCR primers and 250 nM (final concentration) of the TaqManTM probe (Applied Biosystems). Temperature conditions consisted of a step of 30 min at 48 C and a step of 10 min at 95 C, followed by 45 cycles of 60 C for 1 min and 95 C for 15 s. Data were collected during each extension phase of the PCR reaction and analyzed using a software package (Applied Biosystems). Threshold cycles were determined for each gene. 2.7. Data analysis For serum chemistry data, group mean SEM are presented. Differences across different treatment groups were assessed using a one-way ANOVA followed by a Dunnett’s post-hoc test. A t-test was used to assess differences between two treatment groups. For vascular function, ACh and SNP-induced relaxation were calculated as the % relaxation of the PE-induced pre-contraction. Differences
in vascular function were determined using a two-way ANOVA, followed by a Bonferroni post-hoc test. 3. Results 3.1. Effect of uremia and VDRM treatment on blood chemistry and endothelial function As shown in Table 1, the serum creatinine and BUN levels were significantly elevated in 5/6 nephrectomized (NX) rats compared to sham rats on both Day 0 (6 weeks after surgery) and Day 13 (8 weeks after surgery), indicating established uremia. The three VDRMs had no significant effect on either BUN or creatinine (vs. Day 0) except that VS-105 at 0.16 mg/kg significantly reduced serum BUN levels. Serum phosphorus/phosphate (Pi) and calcium (Ca) levels were not significantly different in 5/6 NX rats vs. sham, and the compounds had no significant effect on the Pi and Ca levels except that alfacalcidol at 0.08 mg/kg significantly elevated serum Ca levels, and paricalcitol at 0.08 mg/kg exhibited a modest hypercalcemic effect. Serum PTH levels were significantly elevated in 5/6 NX rats treated with vehicle, and were effectively suppressed by the three compounds at all tested doses. Fig. 1A shows that the maximal aortic relaxation response to acetylcholine was significantly reduced in the aorta from 5/6 NX vehicle-treated rats ( 62.1 4.6% in NX vs. 87.0 1.3% in sham rats), indicating compromised endothelial function. Fig. 1B shows that the aortic relaxation produced by sodium nitroprusside (SNP, an endothelium-independent vasodilator) was similar between the sham and 5/6 NX rats, indicating that the smooth muscle relaxation response is functional. Fig. 1A demonstrates that paricalcitol after two weeks of treatment produced a modest, yet dose-dependent improvement in acetylcholine-induced (endothelium-dependent) aortic relaxation. The maximal relaxation response to acetylcholine with 0.04 and 0.08 mg/kg of paricalcitol was at 68.7 2.2% and 71.0 2.8%, respectively. Consistent with our previous report [14], paricalcitol had no effect on SNP-induced relaxation (Fig. 1B) As a comparison, Fig. 2A shows that 0.04 mg/kg of alfacalcidol after 2-weeks of treatment exhibited a modest effect on the maximal relaxation response to acetylcholine ( 69.8 2.4%), while 0.02 mg/kg of alfacalcidol showed minimal effects. A higher dose (0.08 mg/kg) that induced hypercalcemia was then tested, which further improved the maximal relaxation response to acetylcholine at 76.8 14.1% (vs. the maximal aortic relaxation response to acetylcholine in sham rats at 87.0 1.3%, and in
Table 1 Effects of VDRMs on physiological parameters in sham vs. 5/6 nephrectomized rats. Parameters
Sham 5/6 NX-Vehicle Pari, 0.04 mg/kg Pari, 0.08 mg/kg Alfa, 0.02 mg/kg Alfa, 0.04 mg/kg Alfa, 0.08 mg/kg VS-105, 0.004 mg/kg VS-105 0.01 mg/kg VS-105 0.16 mg/kg
Creatinine (mg/dL)
BUN (mg/dL)
Serum Pi (mg/dL)
Serum Ca (mg/dL)
Serum PTH (pg/mL)
Day 0
Day 13
Day 0
Day 13
Day 0
Day 13
Day 0
Day 13
Day 0
Day 13
0.32 0.02 0.93 0.04a 0.76 0.05a 0.76 0.04a 0.81 0.02a 0.78 0.03a 0.76 0.06a 0.90 0.02a
0.33 0.01 1.05 0.08a 0.86 0.05a 0.90 0.04a,b 0.88 0.03a 0.87 0.04a 0.89 0.11a 0.93 0.03a
15.7 0.5 41.1 1.7a 38.2 2.7a 42.4 2.2a 44.0 2.0a 40.8 1.9a 44.1 3.2a 34.2 1.3a
15.2 0.5 43.9 2.5a 39.8 2.9a 42.0 2.3a 39.8 2.4a 38.8 2.1a 43.7 7.8a 33.0 1.6a
7.5 0.3 7.9 0.3 7.0 0.5 7.2 0.2 8.2 0.2 7.6 0.2 8.2 0.1 7.7 0.8
8.1 0.4 8.1 0.6 6.7 0.8 7.9 0.3 8.4 0.9 7.7 0.3 8.5 0.2 8.3 0.1
9.8 0.2 10.3 0.2 10.3 0.4 9.8 0.3 9.9 0.3 10.2 0.2 10.7 0.1 10.4 0.1
9.9 0.4 10.4 0.7 10.5 0.4 10.9 0.3b 9.3 0.3 10.7 0.2 12.3 0.7b 10.3 0.1
109 8 415 38 379 8 455 43 388 18 460 46 516 66 324 28
126 11 482 40 92 6d 117 41d 98 10d 163 42d 54 30d 216 23b
0.82 0.04a
0.82 0.04a
34.0 1.5a
30.8 1.7a
7.5 0.2
7.3 0.2
9.9 0.1
10.0 0.2
361 22
137 9d
0.92 0.03a
0.93 0.02a
37.8 1.4a
32.3 1.2a,c
7.5 0.2
7.1 0.1
10.6 0.1
10.0 0.1
386 24
96 8d
Rats were treated as in Section 2.1. Data presented are mean SEM (n = 8–12 per group). Pari: paricalcitol; Alfa: alfacalcidol. a p < 0.001 vs. sham Day 0. b p < 0.05 vs. same group Day 0. c p < 0.01 vs. same group Day 0. d p < 0.001 vs. same group Day 0.
J.R. Wu-Wong et al. / Journal of Steroid Biochemistry & Molecular Biology 148 (2015) 202–209
[(Fig._1)TD$IG]
(B)
0 ***
% Relaxation
-25
***
***
*** *** * ***
-50
-75
-100 -10
Sham 5/6 NX Vehicle Pari-0.04 Pari-0.08
-9
*** *** *** ### *** *** *** *** *** *** ***
-8 -7 -6 Ach [log, M]
-5
0
-25
% Relaxation
(A)
-50
-75
-100 -10
-4
205
Sham 5/6 NX Vehicle Pari-0.04 Pari-0.08
-9
-8
-7
-6
-5
-4
SNP [log, M]
Fig. 1. Effect of paricalcitol on aortic relaxation in 5/6 nephrectomized rats. Sham and 5/6 NX rats were treated with vehicle or paricalcitol at indicated doses as described in Section 2.1. Aortic rings were pre-contracted with phenylephrine (PE, 3 mM), and the endothelium-dependent vasodilator acetylcholine (Ach) was added (doses as shown) at 3–5 min intervals. Afterwards, aortic rings were pre-contracted with PE (3 mM) and subsequently treated with the endothelium-independent vasodilator sodium nitroprusside (SNP, doses as shown) at 3–5 min intervals. (A) Acetylcholine-evoked relaxation. (B) SNP-evoked relaxation. ###p < 0.001 vs. sham (at all Ach doses); *p < 0.05, **p < 0.01, ***p < 0.001 vs. NX-vehicle at the indicated Ach dose.
[(Fig._2)TD$IG] 0
(B)
-25 *** -50 -75 -100 -10
Sham 5/6 NX Vehicle Alfa-0.02 Alfa-0.04 Alfa-0.08
-9
***
*** *** ***
-8 -7 -6 Ach [log, M]
###
*** *** *** *** ***
-5
-4
% Relaxation
% Relaxation
(A)
0
### #
*
-25
###
*** -50 -75 -100 0 -10
Sham 5/6 NX Vehicle VS-105 0.004 g/kg VS-105 0.01 g/kg VS-105 0.16 g/kg
-9
-8
###
* ***
-7 -6 Ach [log, M]
### ###
* ***
*** ***
-5
-4
Fig. 2. Effect of alfacalcidol and VS-105 on aortic relaxation in 5/6 nephrectomized rats. Sham and 5/6 NX rats were treated with vehicle or test agents at indicated doses as described in Section 2.1. Aortic rings were pre-contracted with phenylephrine (PE, 3 mM), and the endothelium-dependent vasodilator acetylcholine was added as in Fig. 1. Alfacalcidol and VS-105 were tested in two independent studies using two different groups of 5/6 NX rats. (A) Alfacalcidol. ###p < 0.001 at all Ach doses; ***p < 0.001 vs. NXvehicle at the indicated Ach dose. (B) VS-105. #p < 0.05, ###p < 0.001 vs. sham at the indicated Ach dose; *p < 0.05, ***p < 0.001 vs. NX-vehicle at the indicated Ach dose.
vehicle-treated rats at 62.1 4.6%). Alfacalcidol had no significant effects on SNP-induced relaxation (data not shown). Consistent with our previous report [15], Fig. 2B demonstrates that, VS-105 (i. p., 3/week, for 2 weeks) exerted a dose-dependent improvement in acetylcholine-induced aortic relaxation. The 0.16 mg/kg dose normalized acetylcholine-induced aortic relaxation, while the low dose at 0.004 mg/kg did not exhibit a significant effect. The maximal relaxation response to acetylcholine was at 54.3 8.2% and 78.2 12.9% for 0.01 and 0.16 mg/kg of VS-105, respectively (vs. the maximal aortic relaxation response to acetylcholine at 78.5 5.8% in sham rats, and at 34.5 7.4% in vehicle-treated rats). VS-105 also had no significant effect on SNP-induced endothelial-independent relaxation (data not shown).
3.2. Effects of uremia and VDRMs on aortic gene expression via DNA microarray analysis The hierarchical clustering of genes (with the expression value of each gene in each sample) suggests that uremia exerts a significant effect on aortic gene expression, and all three compounds at the tested dose were able to normalize a subset of genes affected by uremia (see Supplementary Fig. S2). Using thresholds for selecting significant genes at 1.5-fold or 1.5-fold of change and p 0.01, there were 234 genes found to exhibit significant changes in 5/6 NX uremic rats (vs. sham). When the selection criteria were further narrowed down to be at 2-fold or 2-fold of change and p 0.01, 89 genes were tabulated to
Table 2 Aortic genes differentially regulated by uremia and three VDRMs. NX onlya (vs. sham)
NX + paricalcitol (vs. sham)
NX + alfacalcidol (vs. sham)
NX + VS-105 (vs. sham)
89 genes affected (p 0.01, 2-fold) Up-regulated: 68 genes. Down-regulated: 21 genes. –
56 genes affected (p 0.01, 2-fold). Up-regulated: 42 genes. Down-regulated: 14 genes. 33 genes normalized (vs. NX).
46 genes affected (p 0.01, 2-fold). Up-regulated: 35 genes. Down-regulated: 11 genes. 43 genes normalized (vs. NX).
46 genes affected (p 0.01, 2-fold). Up-regulated: 28 genes. Down-regulated: 18 genes. 43 genes normalized (vs. NX)
a
NX only: 5/6 nephrectomized rats treated with vehicle.
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Table 3 Gene ontology analysis of aortic genes affected by uremia and VDRM treatment. Category
Term
Count
p-Value
Benjamini
Normalized by VDRMs
KEGG_PATHWAY INTERPRO KEGG_PATHWAY KEGG_PATHWAY GOTERM_BP_FAT SP_PIR_KEYWORDS GOTERM_BP_FAT KEGG_PATHWAY INTERPRO INTERPRO GOTERM_CC_FAT GOTERM_CC_FAT GOTERM_BP_FAT KEGG_PATHWAY INTERPRO INTERPRO KEGG_PATHWAY
Type I diabetes mellitus MHC class I, alpha chain, C-terminal Graft vs. host disease Allograft rejection Immune response MHC I Antigen processing and presentation of peptide antigen via MHC class I Antigen processing and presentation MHC class I, alpha chain, alpha1 and alpha2 MHC class I-like antigen recognition MHC class I protein complex MHC protein complex Antigen processing and presentation of peptide antigen Autoimmune thyroid disease Immunoglobulin C1-set Immunoglobulin/major histocompatibility complex, conserved site Cell adhesion molecules (CAMs)
10 7 9 9 16 7 7 10 8 8 8 8 7 8 8 8 10
3.2E-11 1.2E-10 3.2E-10 4.4E-10 8.1E-10 8.9E-10 9.8E-10 1.1E-9 1.4E-9 3.1E-9 4.8E-9 2.1E-8 3.0E-8 4.4E-8 1.1E-7 1.1E-7 1.1E-7
1.8E-9 1.9E-8 8.7E-9 8.0E-9 8.9E-7 1.4E-7 5.4E-7 1.5E-8 1.1E-7 1.6E-7 7.7E-7 1.7E-6 1.1E-5 4.9E-7 4.4E-6 4.4E-6 1.0E-6
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
have significant changes in 5/6 NX uremic rats (vs. sham) as summarized in Table 2. Out of these 89 genes, 68 genes were up-regulated and 21 genes were down-regulated. When the data set was analyzed using DAVID (http://david.abcc.ncifcrf.gov/) to assess the general effects of uremia, a number of functionally related clusters were revealed. The top 18 gene ontology categories are shown in Table 3. Among the 89 genes affected by uremia, paricalcitol at 0.08 mg/kg normalized 33 genes, alfacalcidol at 0.04 mg/kg normalized 43 genes, and VS-105 at 0.01 mg/kg also normalized 43 genes (Table 2). There were 22 genes normalized by all three compounds (based on p value) as listed in Table 4. Four genes were normalized by alfacalcidol alone and four genes were normalized by VS-105 alone (based on p value) as listed in Table 5. When the gene data set normalized by all three VDRMs was analyzed using DAVID (http://david.abcc.ncifcrf.gov/), the functionally related clusters were similar to those shown in Table 3, suggesting that VDRM treatment was able to negate the uremic effect.
Table 4 Aortic genes that are normalized by all three VDRMs. Systematic name
ID
NX
NM_001164656 NM_021693 ENSRNOT00000001579 NM_019328 ENSRNOT00000036995 NM_001031642 NM_021836 ENSRNOT00000024094 NM_212504 NM_138525 NM_138541 NM_019287 NM_013183 NM_147206 NM_024391 NM_053977 NM_012975 NM_001107015 NM_013068 TC595358 NM_012738 NM_012737 NM_031669
Fcgbpl1 2.29 Sik1 2.36 Nr4a2 2.65 Adamtsl2 2.69 Serpinb1a 2.69 Junb 3.23 Sct 4.48 Hspa1b 5.26 Mucdhl 5.88 Epcam 6.01 Apob 6.61 Mep1b 7.34 Cyp3a9 8.41 Hsd17b2 9.46 Cdh17 10.48 Lgals4 10.72 Tm4sf5 11.14 Fabp2 11.43 TC595358 13.78 Apoa1 14.97 Apoa4 18.82 Prap1 29.55
Alfa 1.06 1.10 1.55 1.27 1.11 2.69 1.98 3.04 1.34 1.22 1.88 1.45 1.57 1.37 1.46 1.22 1.87 2.58 1.27 2.50 3.31 4.13
Pari 1.01 1.12 1.60 1.78 1.74 2.76 1.99 1.51 1.21 1.53 1.00 1.91 2.22 2.02 1.76 2.65 2.05 1.10 1.33 1.08 1.24 1.28
VS-105 1.20 1.18 1.02 1.64 1.88 1.65 1.87 1.28 1.27 3.04 1.17 2.89 3.08 2.73 2.34 2.38 1.30 1.69 1.37 1.56 1.86 1.82
Note: “Normalization” was based on p value changing from 0.01. NX: 5/6 nephrectomized rats treated with vehicle. Alfa: alfacalcidol; Pari: paricalcitol.
3.3. Confirmation of gene microarray results by real-time RT-PCR As a validation of the results, we employed real-time RT-PCR to examine the expression of 5 selected genes (Abra, NM_175844, actin-binding Rho activating protein; Apoa4, NM_012737, apolipoprotein A-IV; Fabp2, NM_013068, fatty acid binding protein 2; Hsd17b2, NM_024391; hydroxysteroid (17-beta) dehydrogenase 2; Hspa1b, NM_212504, heat shock 70 kDa protein 1B). Fig. 3A shows that the expression level of Abra was elevated in 5/6 NX rats, which was further increased in the paricalcitol and alfacalcidol groups, but not significantly changed in the VS-105 group. Consistent with the microarray analysis, Fig. 3B shows that Apoa4 was elevated in 5/6 NX rats; paricalcitol and VS-105 effectively reduced Apoa4 to the sham level, while alfacalcidol had lesser effects. The Q-PCR results confirmed that Fabp2 was elevated in 5/6 NX rats; all three compounds significantly reduced Fabp2, but alfacalcidol had lesser effects (Fig. 3C). Fig. 3D shows that the expression level of Hsd17b2 was low, but the results are consistent with the microarray analysis that Hsd17b2 was elevated in 5/6 NX rats. Paricalcitol and VS-105 reduced Hsd17b2 to the sham level, while alfacalcidol had lesser effects. Fig. 3E shows that all three compounds at the tested doses reduced the expression of Hspa1b with the most effect observed in the VS-105 group. 4. Discussion We have demonstrated in this report that alfacalcidol, paricalcitol and VS-105 all improve endothelial function in 5/6 NX rats in a dose dependent manner independent of their effects on serum calcium and PTH, but alfacalcidol at nonTable 5 Aortic genes that are normalized by alfacalcidol alone (top 4 genes) or VS-105 alone (bottom five genes). Systematic name
ID
NX
Alfa
Pari
VS-105
NM_134371 NM_133298 NM_001031652 XM_342027
Trpm8 Gpnmb St6galnac2 Ms4a4a
5.07 2.27 2.39 2.83
2.22 1.81 1.39 1.60
5.69 2.57 2.11 2.70
6.44 2.74 3.14 2.97
NM_012797 NM_012620 NM_001108621 NM_175844 ENSRNOT00000001953
Id1 Serpine1 Fam180a Abra Srcrb4d
2.53 4.47 2.11 5.94 5.07
2.70 4.05 2.17 5.83 3.87
2.13 3.82 2.30 4.66 2.19
2.00 2.80 1.87 2.30 1.19
Note: “Normalization” was based on p value changing from 0.01. NX: 5/6 nephrectomized rats treated with vehicle. Alfa: alfacalcidol; Pari: paricalcitol.
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hypercalcemic doses exhibits less of an effect on endothelial function when compared with paricalcitol and VS-105. The current study shows that PTH suppression is not necessarily linked to endothelial functional improvement in uremic rats since all three VDRMs at the tested doses suppress PTH effectively and to similar levels, but they exhibited different effects on endothelial function. Previously we have also shown that cinacalcet, a calcimimetic, suppresses PTH effectively, but does not improve endothelial function [14]. These observations seem consistent with clinical observations in the past few years that the survival and cardiovascular benefit of vitamin D analog therapy in CKD patients is observed across different serum PTH levels and different VDRMs may exhibit differential effects [18–33]. However, it is worth noting that others have shown that elevated PTH may play a role in various cardiovascular disorders including abnormal vasodilation [34]. Previously Nilsson et al. have also reported that, in patients with primary hyperparathyroidism, parathyroidectomy significantly improved endothelial vasodilatory function [35]. Data from the current study suggest that changes in serum calcium do not seem to affect endothelial function, which contrasts with the observation made by Jolma et al. [36] that a high calcium diet, leading to increased serum calcium, enhanced the resistance to artery relaxation in 5/6 NX rats. In this study alfacalcidol at 0.08 mg/kg raised serum calcium significantly, yet its effect on improving endothelial function was greater than the effect at lower doses that did not affect serum calcium, suggesting that the higher the VDRM dose, the greater is its effect on endothelial function. At the same time, it is important to consider that hypercalcemia interferes with numerous physiological functions such as bone formation, hormone release, muscle contraction, and nerve and
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brain functions. Moreover, extreme and/or persistent hypercalcemia can be lethal. Therefore, any degree of hypercalcemia is a condition to be avoided. In clinical setting, serum calcium is frequently monitored and VDRM doses are also frequently titrated in order to achieve PTH suppression without raising serum calcium. Our results suggest that a VDRM with a wider therapeutic index that can be administered at high doses without causing hypercalcemia may exhibit a greater degree of beneficial cardiovascular effects. In our previous study testing the effect of VDRMs on modulating gene expression in human coronary artery smooth muscle cells [37,38], we observed that a large cluster of genes involved in cell differentiation/proliferation were modulated when proliferating smooth muscle cells were treated by VDRMs. In addition, several cardiovascular-related genes were also affected by VDRMs. In this current study, we observed that uremia exerts a detrimental impact on aortic gene expression in 5/6 NX rats. Gene ontology analysis reveals that many of the altered genes are linked to the immune system, which is mostly corrected by VDRM treatment. When examined in more detail, several genes linked to oxidative stress, inflammation, metabolic syndrome, and hormone functions including Apoa4, FABP2, Hsd17b2, Abra, and Hspa1b were identified. Previously Apoa4 (NM_012737; apolipoprotein A-IV) was known to be involved in triglyceride-rich lipoprotein metabolism and reverse cholesterol transport [39]. More recently, it has been suggested that Apoa4 may modulate oxidative stress [40] and play a role in coronary artery calcification [41]. FABP2 (NM_013068; fatty acid binding protein 2), known to regulate the intracellular transport of long-chain fatty acids and their acyl-CoA esters, belongs to the family of fatty acid binding proteins, of which
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Abra/GAPDH, ratio
0.03
##
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Fig. 3. Effects of VDRMs on mRNA expression of selected genes. RNA was isolated from aorta in sham, 5/6 NX rats treated with vehicle, and 5/6 NX rats treated with paricalcitol (0.08 mg/kg), alfacalcidol (0.04 mg/kg), or VS-105 (0.01 mg/kg). Each group consisted of 8–12 aortic RNA samples (one sample per rat). The mRNA levels of target genes were analyzed by real-time RT-PCR. GADPH was used for normalization. (A) Abra. (B) Apoa4. (C) Fabp2. (D) Hsd17b2. (E) Hspa1b. Statistical analysis was performed by unpaired t-test. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. sham; *p < 0.05, **p < 0.01, ***p < 0.001 vs. NX-vehicle. Pari: paricalcitol. Alfa: alfacalcidol.
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[(Fig._4)TD$IG]
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. jsbmb.2014.12.002. References
Fig. 4. Possible mechanisms for the effects of uremia and VDRM treatment in aortic endothelial function. Uremia is associated with inflammation, oxidative stress, abnormal metabolic pathways, and de-regulated hormone functions, leading to endothelial dysfunction, which is largely reversed by VDRM treatment.
several are associated with the metabolic syndrome [42]. FABP2 A54T polymorphism has been linked to impaired glucose tolerance, obesity, altered plasma lipids and lipoproteins [43]. Hsd17b2 (NM_024391; hydroxysteroid (17-beta) dehydrogenase 2), a member in the family of metabolic and regulatory enzymes [44], is known to regulate the biological activity of steroid hormones, including estrogen and androgens [45]. Abra (NM_175844; actin-binding Rho activating protein) may act as an activator of serum response factor (SRF)-dependent transcription through a mechanism involving RhoA and actin polymerization [46]. Targeted deletion of Abra in CL57BL/6 mice led to impaired arteriogenesis [47]. Last but not least, Hspa1b (NM_212504; heat shock 70 kDa protein 1B), a member of the heat shock protein 70 family, may be involved in the ubiquitinproteasome pathway through interaction with the AU-rich element RNA-binding protein 1 [48]. It has been shown that Hspa1b polymorphism is linked to familial forms of inflammatory dilated cardiomyopathy [49]. Taken together, data from the current study suggest that uremia significantly impacts several different mechanisms involved in the cardiovascular system including oxidative stress, inflammation, metabolic pathways and hormone functions, leading to de-regulation of numerous genes, which is largely reversed by VDRM treatment (Fig. 4). Interestingly, even among the five selected genes, data from real-time Q-PCR show that alfacalcidol at 0.04 mg/kg is less effective than VS-105 at 0.01 mg/kg although both compounds at the tested doses suppress serum PTH in a similar manner. In summary, VDR activation by alfacalcidol, paricalcitol and VS-105 significantly improve endothelial function in 5/6 NX uremic rat in a dose-dependent manner. More importantly, the effect of VDRMs on endothelial function is independent of PTH suppression. The results suggest that for VDRM therapy, optimal cardiovascular benefit may be best achieved at higher doses using a less hypercalcemic VDRM. Conflict of interest Chen and Wu-Wong work for Vidasym. Acknowledgements We are in debt to Dr. Jerry L. Wessale for his comments and suggestions.
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