Vitamin D Receptor Activators Can Protect against Vascular Calcification
Suresh Mathew*,
Richard J. Lund,
Lala R. Chaudhary*,
Theresa Geurs* and
Keith A. Hruska*,
Renal Division, Departments of * Pediatrics and Medicine, Washington University School of Medicine, St. Louis, Missouri; and Renal Division, Department of Medicine, Creighton University, Omaha, Nebraska
Correspondence: Dr. Keith A. Hruska, Department of Pediatrics, Campus Box 8208, 5th Fl MPRB, 660 S. Euclid Avenue, St. Louis, MO 63110. Phone: 314-286-2772; Fax: 314-286-2894; E-mail: hruska_k{at}wustl.edu
Received for publication August 15, 2007.
Accepted for publication March 24, 2008.
An apparent conflict exists between observational studies thatsuggest that vitamin D receptor (VDR) activators provide a survivaladvantage for patients with ESRD and other studies that suggestthat they cause vascular calcification. In an effort to explainthis discrepancy, we studied the effects of the VDR activatorscalcitriol and paricalcitol on aortic calcification in a mousemodel of chronic kidney disease (CKD)-stimulated atheroscleroticcardiovascular mineralization. At dosages sufficient to correctsecondary hyperparathyroidism, calcitriol and paricalcitol wereprotective against aortic calcification, but higher dosagesstimulated aortic calcification. At protective dosages, theVDR activators reduced osteoblastic gene expression in the aorta,which is normally increased in CKD, perhaps explaining thisinhibition of aortic calcification. Interpreting the resultsobtained using this model, however, is complicated by the adynamicbone disorder; both calcitriol and paricalcitol stimulated osteoblastsurfaces and rates of bone formation. Therefore, the skeletalactions of the VDR activators may have contributed to theirprotection against aortic calcification. We conclude that low,clinically relevant dosages of calcitriol and paricalcitol mayprotect against CKD-stimulated vascular calcification.
Observational studies have shown that administration of vitaminD receptor (VDR) activators in end-stage kidney disease (ESKD)increases survival compared with untreated dialysis patients.1,2Furthermore, there is an additional survival advantage betweentherapy with the native VDR activator, calcitriol, and paricalcitolfavoring the latter.3 These studies are difficult to understandbecause they are matched by other studies demonstrating thathyperphosphatemia and vascular calcification (VC) are mortalityrisk factors in ESKD and chronic kidney disease (CKD).4–7Both hyperphosphatemia8,9 and VC10–15 are stimulated byVDR activators; therefore, the mechanism of the survival advantageprovided by VDR activators is unknown, and the situation isunclear.
VDR activators are agents approved for the indication of secondaryhyperparathyroidism in CKD. One of the most important difficultiesin the clinical management of CKD and ESKD is determining howmuch suppression of parathyroid hormone (PTH) is ideal. Thereis a balance between VDR activator–induced PTH suppressionand stimulation of intestinal calcium (Ca) and phosphate absorptionthat may limit administration of the drugs as a result of elevatedserum phosphorus (Pi) or Ca levels. Elevations in serum Pi orCa produced by VDR activators may participate in their reputedrole of stimulating VC.13,14 In addition, suppression of PTHlevels to normal ranges in CKD/ESKD will result in adynamicbone disorder (ABD),16,17 and the actions of vitamin D analogsin ABD are not understood. In fact, the finding of ABD whenPTH levels are suppressed during treatment with calcitriol andits analogs has led to incrimination of the VDR activators ascausative agents in the pathogenesis of ABD.16,18 ABD is especiallyassociated with VC in ESKD,19 and avoiding this form of renalosteodystrophy may be very important.20,21
We have discovered that ABD was directly stimulated by CKD inmice when serum Pi, Ca, and PTH levels were maintained normal.22In this model, Pi intake was reduced in proportion to the reductionin GFR, and calcitriol (20 ng/kg three times a week intraperitoneally)was supplemented to avoid osteomalacia as a result of phosphaterestriction. Induction of ABD by CKD has been confirmed by otherinvestigators using thyroparathyroidectomized five-sixths nephrectomizedrats replaced with thyroid hormone and PTH.23 Human studiesof CKD are consistent with a direct effect of renal injury onbone formation.24,25 Thus, CKD directly inhibits bone formation,and secondary hyperparathyroidism is a means of restoring boneformation rates; however, this adaptation is disease causingbecause of lack of feedback control and greater stimulationof bone resorption compared with the increase in bone formationderiving from increased PTH levels.26,27 Whenever PTH levelsare suppressed below values approximately recommended by theKidney Disease Outcomes Quality Initiative (KDOQI) guidelines,28in CKD and ESKD, ABD will resurface.17
To investigate the issue of VDR activators on the relationshipbetween the skeleton and VC, we turned to our animal model ofVC and ABD in mice with CKD.29,30 The model uses renal ablationin the atherosclerotic LDL receptor–deficient (LDLR–/–)mouse fed high-fat Western diets (40% of calories from fat)to produce CKD. CKD markedly stimulated VC in these animals,and it produced hyperphosphatemia as a result of lack of renalexcretion and ABD.30 We have shown that treatment of hyperphosphatemiadiminishes VC in this model.31
The LDLR–/– high fat–fed mouse with CKD hasABD as a result of additive inhibition of skeletal anabolismfrom high-fat feeding, type 2 diabetes, and CKD. This is exactlywhat is observed in clinical medicine in patients with type2 diabetes and CKD/ESKD.32,33 These patients have marked increasesin the tendency for VC to develop associated with CKD just asour LDLR–/– mice with CKD do.34,35 We have linkedCKD stimulation of VC in this model to the development of ABD.30Thus, our animal model is a model of the ABD complicating CKD,and it is an excellent opportunity to test the effects of VDRactivators on the skeleton and the vasculature.
Here we report studies of therapy with either calcitriol orparicalcitol performed as a direct comparison of their actionson development of VC and the ABD in the LDLR–/–high fat–fed CKD model described. We found that both calcitriol(10 and 20 ng/kg) and paricalcitol (50 and 100 ng/kg) administeredthree times a week intraperitoneally were protective againstVC. The dosages of VDR activators were sufficient to producethe effects for which they are clinically approved, PTH suppressionin CKD. The mechanism of VDR activation in inhibiting aorticcalcification seemed to be inhibition of osteoblastic gene expressionin the aorta. Secondarily, we found that paricalcitol and calcitriolstimulated osteoblast function and improved bone turnover inABD. These skeletal actions may have also contributed to theprotective actions of the VDR activators on VC by increasingdeposition of Ca and Pi in orthotropic sites.
Comparative Studies of Calcitriol and Paricalcitol in the LDLR–/– High Fat–Fed CKD Model: VC
These studies were designed as a comparative treatment trialin a translational animal model of CKD-stimulated VC. The animalmodel is the LDLR–/– mouse that was shown to developcalcification of cardiac valves and atherosclerotic plaqueswhen fed a high-fat diet (sham high fat; Figure 1) in agreementwith previous studies.29,36 Furthermore, we previously demonstratedstimulation of atherosclerotic plaque–associated calcificationby CKD in this model.29 In this study, CKD was induced by renalablation at 12 wk of age, and at 22 wk of age (baseline), treatmentwas begun with the VDR activators. The experimental groups wereanalyzed at 28 wk of age. Aortic Ca content was measured asdescribed in the Concise Methods section. In CKD high fat–fedmice receiving the vehicle for paricalcitol (untreated), therewas a significant increase in aortic Ca accumulation inducedby CKD (Figure 1). Previous studies29,30,37 characterized theincrease in aortic Ca produced by induction of CKD in this modelas an intensification of atherosclerotic plaque neointimal calcification.Those results were confirmed in these studies. In addition,there was no evidence of Mönckeberg's medial arterial sclerosisstimulated by CKD or the VDR activators in this study.
Figure 1. Effects of CKD and VDR activators on aortic Ca levels. High-fat feeding of LDLR–/– mice (sham high fat) increased aortic calcification. CKD induced at 12 wk of age stimulated aortic calcification in the group killed at 28 wk of age (untreated). Compared with the untreated group, both calcitriol 20 ng/kg intraperitoneally three times per week and paricalcitol 100 ng/kg intraperitoneally three times per week administered from 22 to 28 wk of age decreased aortic Ca accumulation. The levels of aortic Ca were not different from the baseline CKD group studied at 22 wk, indicating reduction in aortic Ca accumulation. Paricalcitol, 400 ng/kg intraperitoneally three times per week increased aortic Ca accumulation compared with the untreated group.
The effects of treatment with either calcitriol or paricalcitolon aortic Ca content in 28-wk-old mice are shown in Figure 1.There was a trend for each of the clinically relevant dosagesof calcitriol and paricalcitol to reduce neointimal vascularCa content. The trends exhibited dosage dependence, and theanimals that were treated with 20 ng/kg calcitriol and the animalsthat were treated with 100 ng/kg paricalcitol had significantlyless vascular Ca than the uremic high fat–fed vehicle-treatedanimals (Figure 1). A clinically relevant dosage of the VDRactivators was defined as dosages close to the threshold dosagefor the VDR activator indication, suppression of PTH levelsin CKD (Table 1).
Table 1. Biochemical parameters in the various groups of animals
Previous reports demonstrated stimulation of VC by vitamin Danalogs.10,12,15 The actions of the VDR activators on aorticcalcification reported here are not in disagreement with thesereports, because when we used a dosage of paricalcitol (paricalcitol400 ng/kg) equivalent to the VDR activator dosages used in theprevious reports; we also observed stimulation of aortic Cacontent (Figure 1). The stimulation of vascular Ca content inthe group that was treated with 400 ng/kg paricalcitol was dueto increased deposition of vascular Ca in the atheroscleroticplaque.
Aortic Gene Expression
To investigate the mechanism of vascular protection providedby VDR activators, we first analyzed their actions on aorticgene expression. We recently demonstrated that the stimulationof VC by CKD in this model is due to a stimulation of aorticmineralization guided by a bone morphogenic protein-2/4–stimulatedosteoblastic transcription program.37 As shown in Figure 2A,CKD (untreated group) dramatically stimulated aortic osterixexpression. Osterix has been identified as the phosphate-stimulatedosteoblastic transcription factor in studies of human vascularsmooth muscle cells in vitro and as stimulated by CKD and inhibitedby phosphate binders in vivo.31,37 Aortic expression of Msx2(Figure 2B), identified by Cheng et al. 36,38 to be stimulatedin LDLR–/– mice equivalent of our sham high-fatgroup, was increased in the untreated group over the levelsin the sham high-fat group, although the expression levels ofMsx2 remained much lower than those for osterix and CBFA1. TheVDR activators markedly suppressed osterix and Msx2 expression.CBFA1, or Runx2, was also induced by CKD in the untreated group(Figure 2C), and there was a trend for CBFA1 levels to be reducedin the 20-ng/kg calcitriol and the 400-mg/kg paricalcitol groupsthat was NS. Only the reduction in CBFA1 expression seen inthe 50- and the 100-ng/kg paricalcitol groups was significantlyless than the untreated group.
Figure 2. Effects of VDR activators on CKD-induce gene expression in aortas of LDLR–/– high fat–fed mice: (A) osterix. (B) Msx2. (C) CBFA1/RUNX2. (D) osteocalcin. Calcitriol 20 ng/kg and paricalcitol 50 and 100 ng/kg all decreased CKD-induced gene expression. Paricalcitol 400 ng/kg had no effect on CKD-stimulated RUNX2 expression, although it inhibited osteocalcin probably by inhibition of osterix and Msx2 expression. Note that the scale of the y axis was adjusted for the high levels of osteocalcin.
Osteocalcin is the phenotypic marker protein of the osteoblast,and the osteocalcin gene promoter is activated by the osteoblast-specifictranscription factors, especially CBFA1.39–42 Osteocalcinlevels were induced by CKD in the untreated group (Figure 2D).We previously showed by immunohistochemistry the increased osteocalcinprotein in mice of the untreated group, and these studies werenot repeated here.29 These results confirm previous studiesdemonstrating CKD-stimulated heterotopic expression of the osteoblasticphenotype in cells of the aorta in this model.29,37 They arein agreement with numerous studies reporting expression of osteoblasticproteins in the vasculature associated with CKD-stimulated VC.29,43–49Although each of the dosages of the VDR activators decreasedaortic osteocalcin expression, the pattern of effects on expressionmirrored the pattern of expression of CBFA1. The highest dosageof paricalcitol, 400 ng/kg, demonstrated suppression of osterixand Msx2 expression and a loss of effect on CBFA1 expression,perhaps consistent with the stimulation, not inhibition, ofaortic Ca accumulation at this dosage as shown in Figure 1.
Bone Histomorphometry
Because of our previous demonstrations of the role of the skeletonin the VC stimulated by CKD in this model,30,31 we investigatedthe effects of the VDR activators on bone histomorphometry.Distal femoral metaphyseal trabecular bone volume was 10.8%in C57Bl6 wild-type mice (Figure 3). Metaphyseal trabecularbone volume was not significantly different in LDLR–/–mice fed chow diets (Figure 3). High-fat feeding and the inductionof CKD in high fat–fed LDLR–/– mice (untreatedgroup) significantly reduced bone volume compared with shammice that were fed regular chow (Figure 3). While treatmentwith calcitriol had no effect on the osteopenia of the ABD,paricalcitol both 50 and 100 ng/kg restored bone volume in CKDhigh fat–fed LDLR–/– mice to the levels ofwild-type mice (Figure 3). The metaphyseal trabecular bone volumeof the 400-ng/kg paricalcitol group was significantly increasedfrom that of the untreated group but significantly less so thanthe 50-ng/kg paricalcitol group.
Figure 3. Effects of VDR activators on metaphyseal trabecular bone volume (bv/tv) in LDLR–/– high fat–fed mice with CKD and ABD. Bone volume was decreased in the baseline and untreated CKD groups compared with the sham high-fat group. Paricalcitol 50 and 100 ng/kg increased bone volume.
Osteoblast surfaces, as a percentage of bone surface (obs/bs),were 5.5% in C57Bl6 wild-type mice and increased to 9.5% inLDLR–/– sham-operated mice fed regular chow (Figure 4)as we have previously demonstrated.30 High-fat feeding and inductionof CKD significantly reduced osteoblast surface to 4.9 and 4.7%in the CKD baseline and untreated groups, respectively (Figure 4).These findings are compatible with the induction of ABD by CKDin this animal model as previously reported.30 Osteoblast surfaceswere increased by each of the dosages of calcitriol and paricalcitol(Figure 4).
Figure 4. Effects of VDR activators on bone surfaces covered by osteoblasts (obs/bs) in LDLR–/– high fat–fed mice with CKD and ABD. Osteoblast surfaces were decreased in the sham high-fat group and the baseline and untreated CKD groups compared with LDLR–/– chow-fed (sham chow group) animals. Both of the VDR activators increased osteoblast surfaces.
Osteoid volume was 0.07% in the metaphyseal trabecular bonein the untreated group, reflective of the low osteoid volumein the ABD of CKD (Figure 5). Osteoid volume was increased byboth dosages of paricalcitol and the highest calcitriol dosage(20 ng/kg), compatible with increased bone turnover (Figure 5).Why the lowest dosage of calcitriol did not increase osteoidvolume is unclear because it did increase osteoblast surfacesand bone formation rate.
Figure 5. Effects of VDR activators on osteoid volume (ov/tv) in LDLR–/– high fat–fed mice with CKD and ABD. Osteoid volume was decreased in the baseline and untreated CKD groups compared with sham-operated animals. Calcitriol at 20 ng/kg and all dosages of paricalcitol increased osteoid surfaces back to normal levels.
Osteoclast surface as a percentage of bone surface was 2.4%in C57Bl6 wild-type mice and 1.8% in LDLR–/– sham-operatedmice fed regular chow (Figure 6). High-fat feeding did not affectosteoclast surface, and induction of CKD did not affect osteoclastsurface in contrast to its effects on osteoblast surfaces. Thefailure of coordinate downregulation of osteoblasts and osteoclastsis a widely recognized component of ABD in CKD.50 Calcitrioltended to increase osteoclast surfaces further (Figure 6), whereasparicalcitol tended to decrease them, and 50 ng/kg paricalcitolsignificantly reduced osteoclast surface from 2.6% in the baselinegroup to 1.4% (Figure 6).
Figure 6. Effects of VDR activators on bone surfaces covered by osteoclasts (ocs/bs) in LDLR–/– high fat–fed mice with CKD and ABD. Osteoclast surfaces were not affected by the induction of CKD (untreated group) compared with the sham high-fat group. Osteoclast surfaces were significantly reduced in the 50-ng/kg paricalcitol group.
Bone formation rates in mm3/m2 per yr were 0.03 in C57Bl6 wild-typemice, and they were not significantly different in LDLR–/–mice that were sham operated and fed regular chow. Both high-fatfeeding and CKD reduced bone formation rates (Figure 7). Bothcalcitriol and paricalcitol tended to increase bone formationrates, but these effects were less consistent than the increasein osteoblast surfaces. Mineralizing surfaces were 7.6 ±0.9% in sham-operated high fat–fed mice, and these weredecreased by the induction of CKD. Mineralizing surfaces tendedto recover in the paricalcitol-treated animals (Figure 8).
Figure 7. Effects of VDR activators on bone formation rates (bfr/bs) in mm3/m2 per yr in LDLR–/– high fat–fed mice with CKD and ABD. Bone formation rates were decreased in the sham-operated, high fat–fed group and the baseline and untreated CKD groups compared with sham-operated chow-fed animals. Calcitriol at 10 ng/kg and paricalcitol at 100 ng/kg increased bone formation rates.
Figure 8. Effects of VDR activators on mineralizing surfaces (ms/bs). Mineralizing surfaces tended to be decreased in the vehicle and calcitriol CKD animals compared with sham high fat–fed animals. The decrease was significant in the 20-ng/kg calcitriol group. Mineralizing surfaces were significantly increased in the 100-ng/kg paricalcitol group compared with the untreated group.
Serum Chemistries
The serum Pi of C57Bl6 chow-fed mice was 7.7 mg/dl and of LDLR–/–mice fed regular chow was 8.0 mg/dl (Figure 9). High-fat feedingin sham-operated LDLR–/– mice tended to increasethe serum Pi, as we have previously reported. The inductionof CKD by renal ablation in the high fat–fed LDLR–/–mice produced significant hyperphosphatemia with serum Pi levelsof 11.5 and 11.0 mg/dl in the baseline and untreated groups,respectively. The CKD-induced hyperphosphatemia tended to beameliorated in each of the paricalcitol- and calcitriol-treatedgroups (Figure 9), and 20 ng/kg calcitriol, 100 ng/kg paricalcitol,and 400 ng/kg paricalcitol significantly reduced serum Pi levels.
Figure 9. Effects of VDR activators on the serum Pi in LDLR–/– high fat–fed mice with CKD. CKD produced hyperphosphatemia in the baseline (22 wk) and untreated groups (28 wk). The hyperphosphatemia tended to be ameliorated by treatment with the VDR activators, and calcitriol 20 ng/kg and paricalcitol 100 ng/kg significantly reduced serum Pi levels.
The serum Ca was 8.5 mg/dl in the C57Bl6 wild-type mice and8.1 mg/dl in the LDLR–/– chow-fed mice (Figure 10).High-fat feeding tended to decrease the serum Ca to the levelof 7.1 mg/dl. Induction of CKD transiently increased serum Calevels to 10 mg/dl in the baseline CKD group studied at 22 wk.This group also had the highest PTH levels among the groupswith CKD. The increase in the serum Ca was not present in theCKD group (untreated group) studied at 28 wk, and serum Ca levelswere not significantly affected by either calcitriol or paricalcitolcompared with the untreated group (Figure 10).
Figure 10. Effects of VDR activators on the serum Ca in LDLR–/– high fat–fed mice with CKD. A transient increase in the serum Ca was observed in the baseline CKD group that was not present in the untreated group at 28 wk. Serum Ca levels were not different among the various treatment groups.
In Table 1, the blood urea nitrogen (BUN) levels in the variousanimal groups are shown. BUN was 18 mg/dl in the sham-operated,high fat–fed animals and elevated in the CKD groups (range37 to 61 mg/dl). The BUN levels were not significantly differentbetween the CKD groups. The renal function impairment of theCKD animals in this study was equivalent to human stage 3 CKD.
PTH levels were increased by induction of CKD as expected onthe high-fat diet (0.6% Pi; 743 and 443 pg/ml in the baselineand untreated groups, respectively; Table 1). The VDR activatorsdosage-dependently and significantly suppressed PTH levels ateach of the dosages selected for study in this project.
High-fat feeding induced marked hypercholesterolemia in theLDLR–/– mice (sham high fat) as previously reported.30,36Neither CKD nor any of the treatments except the high-dosageparicalcitol (400 ng/kg) had significant effects on the hypercholesterolemia.The CKD high fat–fed mice in this study at 28 wk weremore insulin resistant than the baseline CKD group at 22 wk,manifested as the increase in blood glucose from 124 to 165mg/dl. We previously noted that this model advances from insulinresistance to type 2 diabetes after 22 wk.31 There was no effectof the VDR activators on blood glucose levels. The sham-operated,high fat–fed mice gained more weight than the other groups,and CKD decreased the weight gain induced by the high-fat diet.Neither of the dosages of paricalcitol or calcitriol had aneffect on weight gain.
In the studies reported here, paricalcitol and calcitriol, atdosages just sufficient to decrease PTH levels and thus definedas equivalent to those that are clinically used, tended to decrease,not increase, accumulation of vascular Ca levels from 22 to28 wk in LDLR–/– high fat–fed mice with CKDcompared with vehicle-treated control animals. The reductionin vascular Ca accumulation was significant in mice receivingthe 20-ng/kg calcitriol and the 100-ng/kg paricalcitol dosages,and was associated with reduced expression of osteoblast-specifictranscription factors and one of their target proteins, osteocalcin,in the aortas of treated animals. These results are consistentwith previous studies of aortic calcification in this modelas being due to an osteoblastic transcription program expressedin cells of the atherosclerotic neointima,36,51,52 which isstimulated by CKD.29–31,37 The results are also consistentwith the demonstration of CKD stimulation of neointimal aorticcalcification in the apolipoprotein E–/– mice.53Previous studies31,37 demonstrated that the VC in the LDLR–/–high fat–fed CKD model is reversible, as would be expectedwith mineralization analogous to bone formation as a resultof remodeling, and osteoclasts have been found in calcifiedatherosclerotic vessels.54–57 Thus, the protective actionsof the VDR activators reported here, although impressive andsurprising, were less potent than factors that completely normalizedthe hyperphosphatemia produced by CKD and actually decreasedaortic Ca levels at 28 wk compared with 22-wk baseline animals.31
Previous studies30,31 associating improvement in ABD associatedwith CKD and amelioration of VC called our attention to potentialskeletal actions of the VDR activators on ABD. Paricalcitolincreased bone volume, osteoblast surfaces, and osteoid volumeand decreased osteoclast surfaces. Calcitriol increased osteoblastsurfaces and less consistently increased osteoid volume andbone formation rates. Calcitriol did not affect osteoclast surfacescompared with the baseline and untreated CKD groups. In summary,it is possible that the reductions in vascular Ca observed bytreatment with vitamin D analogs of LDLR–/– highfat–fed mice with CKD may have been contributed to secondarilyby positive changes in ABD produced by CKD in these animals.Inconsistencies in the bone histomorphometry data describedin the results prevented us from drawing stronger conclusionsregarding the role of the skeleton in the amelioration of VCstimulated by the VDR activators. The alleviation of osteoclast-mediatedbone resorption by paricalcitol may have produced an increasein bone volume, and this could well contribute to a reductionin the osteopenia produced by ABD in CKD.
The effects of the VDR activators on the skeleton in these studiesmay have contributed to their effects on the serum Pi. In addition,stimulation of fibroblast growth factor 23 production by theVDR activators could have stimulated urinary excretion of Pi,which was not studied in the experiments reported here.
Several previous studies demonstrated that vitamin D analogsstimulate VC.10–12,15 Our results are not in disagreementwith these studies, because when we used higher dosages of paricalcitol,we also demonstrated stimulation of aortic calcification, inagreement with the previously published studies. We do not havedata on the effects of the VDR activators on vascular smoothmuscle cell PTH related protein (PTHrp) expression in contrastto Jono et al.11 What our results indicate is that a biphasicdosage-response curve exists in the effects of VDR activatorson aortic mineralization. Whereas lower dosages are inhibitory,higher dosages are stimulatory. This biphasic response to VDRactivators was observed in the osteoblast transcription factorgene CBFA1/RUNX2 expression in the aortas of the high fat–fedmice with CKD. Whereas 50 and 100 ng/kg paricalcitol inhibitedRUNX2 expression, 400 ng/kg restored RUNX2 expression to theelevated levels found in the untreated high fat–fed CKDmice. These data may relate to the human clinical situation,and they suggest that lower dosages of VDR activators are preferableover very high dosages. In fact, the observational studies1–3demonstrate no dosage dependence in the survival benefit affordedby VDR activators, favoring use of lower dosages.
The studies reported here are the first to analyze therapy ofABD with VDR activators. In agreement with studies of VDR-deficientand 1-hydroxylase–deficient mice,58–60 the VDR activatorsstimulated osteoblast function instead of suppressing it andincreased osteoblast surfaces and bone formation rates. Theseresults are in disagreement with conclusions drawn from theappearance of the ABD when PTH levels were suppressed with VDRactivators.17 An alternative conclusion, that suppression ofhyperparathyroidism uncovers the effect of CKD on skeletal anabolism,would be in agreement with our studies demonstrating this phenomenon.22
The VDR activators are cell differentiation factors, and thevascular smooth muscle from which some cells in the neointimaare derived have VDR.61 The inhibition of osteoblastic factorsin the aorta by VDR activators reported here suggests the needfor further study of vascular smooth muscle phenotype influencedby VDR activators in CKD and characterization of the dosingof VDR activators in VC. These actions could contribute to theiractions to ameliorate hypertension, and they are compatiblewith the observational studies suggesting decreased cardiovascularmortality associated with VDR activator therapy in patientswith ESKD.1–3 They are also compatible with the inverserelationship between calcitriol levels and coronary artery Calevels determined by electron-beam computed tomography.62
Animals and Diets
LDLR–/– mice of both genders in a C57Bl6 backgroundwere purchased from Jackson Laboratory (Bar Harbor, ME) andwere bred in a pathogen-free environment. Animals were weanedat 3 wk to a chow diet (1:1 mixture of Pico Lab rodent chow20 and mouse chow 20, 6.75% calories as fat). At 10 wk, animalswere continued on this chow diet or initiated on a high-cholesterol(0.15%) diet containing 42% calories as fat (product no. TD.88137;Harlan Teklad, Madison WI), 0.6% Pi, 0.6% Ca, and vitamin Dcontent 2.2 IU/g, a diet that has been shown to generate atherosclerosiswith VC in this genetic background. At 12 wk, CKD was inducedas described in the next section. Animals had access to waterad libitum and were maintained according to local and nationalanimal care guidelines. Paricalcitol and calcitriol were providedto us by Abbott (Abbott Park, IL). The paricalcitol and calcitriolwere provided as powder. The powder was dissolved in 100% ethanol(ETOH) and subsequently diluted in 5% ETOH to appropriate concentrations.Xylazine, ketamine, and tetracycline were obtained from Sigma-AldrichCo. (St. Louis, MO). The Washington University Animal Care Committeeapproved the study protocol.
Induction of CKD and Treatment Protocol
A two-step procedure was used to create CKD as described previously.29,30Briefly, electrocautery was applied to the right kidney througha 2-cm flank incision at 10 wk postnatal, followed by left totalnephrectomy through a similar incision 2 wk later. Control animalsreceived sham operations in which the appropriate kidney wasexposed and mobilized but not treated in any other way. StableCKD was established after the two surgical procedures. Afterthe surgical procedures, the 14-wk-old mice were randomizedinto 10 groups. The first was wild-type mice fed a regular diet.This was the normal renal function and diet group. The secondgroup was LDLR–/– mice fed regular chow. This groupserved as the genotype and the control for the diet. The thirdgroup was sham-operated LDLR–/– mice that were feda high-fat diet. This group had normal renal function. Thisgroup served as the control group to determine the effect ofhigh-fat diet in the face of normal renal function. The fourthand fifth groups were LDLR–/– mice that had CKDand were fed high-fat diet and killed at 22 wk, the baselinegroup, or treated with vehicle (5% ETOH) and killed at 28 wk,the fifth or untreated group. These groups were expected todevelop hyperphosphatemia, ABD, and VC. The sixth and seventhgroups were LDLR–/– mice that had CKD and were fedhigh-fat diet and treated with calcitriol 10 or 20 ng/kg threetimes per week by intraperitoneal injections, respectively.These were the first and second therapy groups. The eighth andninth groups were LDLR–/– mice that had CKD andwere fed high-fat diet and treated with paricalcitol 50 or 100ng/kg three times per week, respectively. The tenth group wasLDLR–/– mice that had CKD and were fed high-fatdiet and treated with paricalcitol 400 ng/kg three times perweek. This was the high-dosage group expected to produce signsof toxicity. Once the mice were randomized into groups, theywere allowed to develop calcification from weeks 14 through22 postnatal. Therapy was initiated at 23 wk postnatal and continueduntil week 28 postnatal, at which time the mice were killedunder anesthesia. Intraperitoneal anesthesia (xylazine 13 mg/kgand ketamine 87 mg/kg) was used for all procedures. Saphenousvein blood samples were taken 1 wk after the second surgeryto assess baseline postsurgical renal function. At the timeof killing, blood was taken by intracardiac stab, and the heartand aorta were dissected en block.
Chemical Calcification Quantification
Aorta and hearts were dissected when the mice were killed, andall extraneous tissue was removed by blunt dissection undera dissecting microscope. Tissues were desiccated for 20 to 24h at 60°C, weighed, and crushed to a powder with a pestleand mortar. Ca was eluted in 1 N HCL for 24 h at 4°C. Cacontent of eluate was assayed using a cresolphthalein complexonemethod (Sigma), according to the manufacturer's instructions,and results were corrected for dry tissue weight.
Blood Tests
Serum was analyzed on the day of blood draw for BUN, cholesterol,Ca, glucose, and phosphate by standard autoanalyzer laboratorymethods performed by our animal facility.
Bone Histology and Histomorphometry
Bone formation was determined at the time of killing. All micereceived intraperitoneal tetracycline (5 mg/kg) 7 and 2 d beforebeing killed. Both femurs were dissected at the time of killingand placed in 70% ETOH. The specimens were implanted undecalcifiedin a plastic embedding kit H7000 (Energy Beam Sciences, Agawam,MA). Bones were sectioned longitudinally through frontal planein 5-µm sections with a JB-4 Microtome (Energy Beam Sciences).Tissue was stained with Goldner's trichrome stain for trabecularand cellular analysis. TRAP staining was used to identify osteoclastsand define osteoclast surfaces. Unstained 10-µm sectionswere used for tetracycline-labeled fluorescence analysis. Slideswere examined at x400 magnification using a Leitz microscopeattached to an Osteomeasure Image Analyzer (Osteometrics, Atlanta,GA). Ten contiguous 0.0225-mm2 fields of the distal femur, 150µm proximal to the growth plate, were examined per animal.Primary, derived, and kinetic measures of bone remodeling werecalculated and reported per guidelines of the American Societyof Bone and Mineral Research.63
Reverse Transcription–PCR
RNA was extracted from aortas using RNeasy Mini Kits (Qiagen,Valencia, CA). Total RNA (1 µg) was reverse-transcribedusing iScript cDNA synthesis kit from Bio-Rad (Hercules, CA)according to the manufacturer's instructions. Primers were designedusing Vector NTI software (Invitrogen, Grand Island, NY), andoptimal conditions for each primer pair were determined (Table 2).A Perkin-Elmer DNA Thermal Cycler was used to perform the reaction.After reverse transcription performed as described, real timewas performed using the MX 4000 (Stratagene, La Jolla, CA),SYBR Green from Sigma, and the PCR kit from Invitrogen. Eachreaction was performed in triplicate at 95°C for 45 s, 60°Cfor 30 s, and 60 s at 72°C for 40 cycles. This was followedby a melt cycle, which consisted of stepwise increase in temperaturefrom 72 to 99°C. A single predominant peak was observedin the dissociation curve of each gene, supporting the specificityof the PCR product. Threshold values were set within the exponentialphase of PCR and were used to calculate the expression levelsof the genes of interest. Glyceraldehyde-3-phosphate dehydrogenasewas used as an internal standard and used to normalize the values.A standard curve consisting of the threshold value versus logcDNA dilutions (corresponding to the log copy numbers) was generatedby amplification of serial dilutions of cDNA corresponding toan unknown amount of amplicon. Negative controls were performedby inactivating the reverse transcriptase by boiling for 5 minbefore reverse transcription–PCR to ensure that genomicDNA was not amplified.
Statistical Analysis
Statistical analysis was performed using ANOVA. Differencesbetween groups were assessed post hoc using Fisher least significantdifference method and considered significant at P < 0.05.Data are presented as means ± SEM. Analyses were performedusing Sigma Stat statistical software (Point Richmond, CA).
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Abstract
Introduction
-hydroxylase–deficient mice,58–60 the VDR activators stimulated osteoblast function instead of suppressing it and increased osteoblast surfaces and bone formation rates. These results are in disagreement with conclusions drawn from the appearance of the ABD when PTH levels were suppressed with VDR activators.17 An alternative conclusion, that suppression of hyperparathyroidism uncovers the effect of CKD on skeletal anabolism, would be in agreement with our studies demonstrating this phenomenon.22 
