Effect of Simvastatin in Apolipoprotein E DeficientMice With Surgically Induced Chronic Renal FailureOgnen Ivanovski,* Dorota Szumilak, Thao Nguyen-Khoa, Igor G. Nikolov, Nobuhiko Joki,Nadya Mothu, Julien Maizel, Ralf Westenfeld, Marcus Ketteler, Bernard Lacour, Tilman B. Drüekeand Ziad A. Massy†,‡From the Institut National en Santé et Recherche Médicale Unit 845 (OI, DX, TNK, IGN, NJ, NM, TBD), University Paris V (OI, DX,TNK, IGN, NJ, NM, TBD) and Laboratory of Biochemistry A, Necker Hospital (TNK, BL), Paris and Institut National en Santé etRecherche Médicale ERI-12 and University of Picardie and Amiens University Hospital, Amiens (IGN, JM, ZAM), France, andDepartment of Nephrology and Clinical Immunology, University Hospital Aachen (RW, MK), Aachen, Germany

Purpose: Patients with a surgically reduced renal mass are at increased risk for progressive renal failure, which oftenrequires renal replacement therapy or kidney transplantation. We investigated the effects of simvastatin supplementation onuremia enhanced atherosclerosis and vascular calcification in apoE�/� (apolipoprotein E deficient) mice (Charles RiversLaboratories, Wilmington, Massachusetts) with or without superimposed chronic kidney disease.Materials and Methods: The mice were randomly assigned to 4 groups, including 2 groups with normal renal function(simvastatin vs control in 13 mice) and the other 2 with surgically created chronic kidney disease (simvastatin vs control in18). Simvastatin (100 mg/kg) was administered by daily oral gavage for 4 weeks.Results: Simvastatin treatment did not prevent uremia accelerated atherosclerosis in chronic kidney disease apoE�/� mice,nor did it retard atherosclerosis progression in control nonchronic kidney disease mice. However, aortic plaques in simva-statin treated chronic kidney disease mice showed significantly less calcification than those in controls with chronic kidneydisease (p �0.03). In addition, the increase of aortic nitrotyrosine staining in mice with chronic kidney disease was preventedby simvastatin treatment (p �0.02). Serum total cholesterol was increased to a similar extent in the 2 chronic kidney diseasegroups compared with that in the nonchronic kidney disease groups. The beneficial effect of simvastatin on uremia enhancedvascular calcification in apoE�/� mice with chronic kidney disease was observed despite the absence of changes in uremiaaccelerated atherosclerosis progression, serum total cholesterol levels or osteopontin and alkaline phosphatase expression.Conclusions: Our observation opens the possibility of a cholesterol independent action of statins on vascular calcification viaa decrease in oxidative stress.

Key Words: vascular calcification, surgery, oxidative stress, statins

HMG-CoA (3-hydroxy-3-methyl-glutaryl-CoA reduc-tase) inhibitors, generally called statins, are com-monly used to lower serum total and low density

lipoprotein cholesterol. They have proven efficacy in de-creasing cardiovascular events in the general population.1

However, there remains substantial uncertainty regardingthe effects of statin therapy in patients with establishedCKD. The negative result of the Die Deutsche DiabetesDialyse study comparing the effect of atorvastatin with pla-cebo on cardiovascular outcomes in patients with diabeteson dialysis came as a surprise.2 Atypical cardiovasculardisease in such patients was one of several possible expla-

Submitted for publication July 20, 2007.Supported by a grant from the EGIDE Foundation, Paris, France

(OI, IN) and Toho University funds, Japan (NJ).* Current address: Clinic of Urology, University Clinical Centre

Skopje, Medical Faculty, Skopje, Republic of Macedonia.† Correspondence: INSERM ERI-12, Division of Clinical Pharma-

cology and Nephrology, University of Picardie and Amiens Univer-sity Hospital, Av. René Laennec, F-80054 Amiens, France (tele-phone: � 33 3 2245 5788; FAX: � 33 3 2245 5660; e-mail: massy@u-picardie.fr).

‡ Financial interest and/or other relationship with University ofOxford/Merck Schering Plough.

0022-5347/08/1794-1631/0THE JOURNAL OF UROLOGY®

Copyright © 2008 by AMERICAN UROLOGICAL ASSOCIATION

1631

nations to explain this negative result. Indeed, the mostmarked difference between patients with and without ure-mia is not the size of atherosclerotic plaque but its compo-sition with a marked increase in calcium and collagen con-tent per plaque area.3

Observational studies have suggested a role of low den-sity lipoprotein cholesterol levels in coronary artery calcifi-cation4 and an inhibitory effect of statin therapy.4 However,a recent randomized, prospective trial failed to confirm thatstatin decreases coronary artery calcification in the generalpopulation.5 Thus, the issue remains controversial.

Even in the absence of cholesterol lowering, statins maybe beneficial in the treatment of atherosclerosis due to localanti-inflammatory and antioxidative actions. They may oc-cur via the up-regulation of endothelial nitric oxide synthasewith the increased bioavailability of nitric oxide, a decreasein cellular proliferation, an increase in apoptosis and/or aninterference with local oxidative injury.6 Many of thesepleiotropic statin effects are mediated by their ability toblock the production of isoprenoid intermediates such asfarnesyl or geranylgeranyl pyrophosphates, which appear tobe important in the activation of small guanosine triphos-

phate binding proteins, including Rho, Ras and Rac, through

Vol. 179, 1631-1636, April 2008Printed in U.S.A.

DOI:10.1016/j.juro.2007.11.042

SIMVASTATIN IN APOLIPOPROTEIN E DEFICIENT MICE WITH RENAL FAILURE1632

an isoprenylation process.7 However, the precise involve-ment of such pleiotropic effects in the clinical efficacy ofstatins remains unclear because of the difficulty of separat-ing lipid related vs nonlipid related responses.

In apoE�/� mice hypercholesterolemia and atheroscle-rotic lesions develop spontaneously, similar to those in hu-mans.8 However, a difference vs the clinical setting is that inthis model statins do not decrease serum total cholesterollevels.9 We recently established a new mouse model by in-ducing CKD in the apoE�/� mouse, leading to more severevascular calcification and atherosclerosis than in nonCKDcontrol mice.10 The current study was designed to examinepossible cholesterol lowering independent effects of simva-statin supplementation on CKD enhanced atherosclerosisand vascular calcification in apoE�/� mice with surgicallyinduced chronic renal failure.

MATERIALS AND METHODS

AnimalsWe obtained apoE�/� homozygous mice of each gender andsubsequently bred them at our animal facility. Animals werehoused at a pathogen-free temperature controlled (25C) fa-cility with a strict 12-hour light-dark cycle and they weregiven free access to water and a standard chow diet (HarlanTeklad Global Diets® 2018). The diet contained 18.9% pro-tein, 6% fat, 1.01% calcium, 0.65% phosphorus, a calcium-to-phosphorus ratio of 1.55 and 1.54 IU/gm vitamin D3. Allprocedures were done in accordance with National Insti-tutes of Health guidelines for the care and use of experimen-tal animals (National Institutes of Health Publication No.85-23). After sexual maturation at age 4 weeks the micewere separated by gender and housed in groups of up to 5.

Study ProtocolAt age 8 weeks CKD was induced by a 2-step procedureinvolving electrocoagulation of the surface of the surgicallyexposed right kidney, followed by left total nephrectomythrough a similar contralateral incision 2 weeks later, aspreviously described.10 Control animals underwent shamoperation involving the decapsulation of each kidney.

Blood samples were taken 10 weeks after the secondintervention. Animals in the CKD group with a serum urealevel of more than 20 mM were randomized to 2 subgroups.The 10 mice in subgroup 1 were treated with simvastatin,

TABLE 1. Characterist

Mean � SEMControl Placebo

MeaContro

Body wt (gm):At randomization 28.4 � 1.9 26.At sacrifice 27.9 � 1.8 29

Urea level (mm):At randomization 8.8 � 0.4 8.At sacrifice 9.9 � 0.4#* 8.

Serum calcium (mM) 1.9 � 0.094 2.Serum phosphorus (mM) 2.1 � 0.23 2.Mean arterial pressure (mm Hg) 70 � 3.2 76.Urinary protein/creatinine ratio (gm/mM) 1.7 � 0.8 3.

Six to 10 mice per group but 4 for mean arterial pressure with data analyz* Vs controls at randomization ANOVA p � 0.04.† Vs urea CKD at randomization ANOVA p � 0.006.

‡ Vs controls at randomization ANOVA p � 0.05.

whereas the 8 in subgroup 2 received placebo. Likewisecontrol nonuremic mice were divided into 2 subgroups, in-cluding 7 in subgroup 1 that were treated with simvastatinand 6 in subgroup 2 that received placebo. Simvastatin wasadministered by oral gavage 6 times per week at a dose of100 mg/kg for 4 weeks. Control animals were given PBSvehicle solution only. Pilot experiments were done with ahigher dose of simvastatin (200 mg/kg 6 times per week),which led to high mortality even in nonCKD control ani-mals. Since simvastatin has renal clearance and its dosemust be adapted by kidney failure status, we used lowerdoses of simvastatin than those used in previous experiments.

Biochemical AnalysisBlood samples were obtained from the retrobulbar orbitalplexus at baseline, 10 weeks after nephrectomy and at end ofstudy. Serum urea and total cholesterol concentrations weremeasured using a Hitachi 917 autoanalyzer (Roche, Meylan,France). Serum urea was used to evaluate the degree ofCKD induced in the animals. Serum calcium and phospho-rus concentrations were measured only at sacrifice using aHitachi autoanalyzer, as mentioned. Urine was taken beforesacrifice, and urinary protein and creatinine concentrationwere measured as reported previously.11

Light Microscopy FindingsFollowing the completion of treatment mice were anesthe-tized with intraperitoneal ketamine/xylazine injection (100and 20 mg/kg, respectively) and whole blood was collectedvia cardiac puncture. The kidneys were immediately dis-sected and preserved in 4% acid-formaldehyde solution, thensliced and stained with hematoxylin and eosin for lightmicroscopy evaluation. The aorta and its main brancheswere dissected from the aortic sinus down to the renal ar-teries, as reported previously.11

Quantification of Atherosclerotic LesionsSerial 10 �m sections of the aortic sinus with valves (60 to 80per mouse) were cut in a cryostat, as reported previously.11

Evaluation of the atherosclerotic plaque area of the entireaorta opened longitudinally was made by the en face method11

using the same image analysis system. The extent of athero-sclerosis is expressed as the percent of surface area of theentire aorta covered by lesions. Images were captured on a

mice at end of study

EMastatin

Mean � SEMCKD Placebo

Mean � SEMCKD

SimvastatinCKD/Statin/Interaction

Effect p Value

8 26.2 � 1.9 27.2 � 1.6 NS/NS/NS6 27.5 � 2.3 27.3 � 1.5 NS/NS/NS

4 27.4 � 1.6 27.6 � 1.6 �0.001/NS/NS6† 25.7 � 2.5 23.3 � 1.7‡ �0.001/NS/NS014 2.3 � 0.015 2.3 � 0.018 �0.001/NS/NS09 2.5 � 0.5 1.8 � 0.08 NS/NS/NS2 73.3 � 2.2 70 � 4.0 NS/NS/NS

1.5 � 0.3 1.8 � 0.3 NS/NS/NS

th 2 factors (CKD state/simvastatin and interaction) considered.

ics of

n � Sl Simv

8 � 1.� 1.

9 � 0.3 � 0.2 � 0.1 � 0.2 � 4.4 � 1

ed wi

SIMVASTATIN IN APOLIPOPROTEIN E DEFICIENT MICE WITH RENAL FAILURE 1633

microcomputer equipped with Histolab software (Microvi-sion Instruments, Evry, France) and analyzed by computer-ized image analysis in blinded fashion.

Quantitative and QualitativeEvaluation of Aorta CalcificationWe performed von Kossa staining in cryosections of aortictissue to evaluate calcium deposits in atheromatous plaques.The precision and accuracy of this assay have been reportedpreviously using semi-automated measurement software.10

Quantification ofMonocyte-Macrophage Infiltration,Collagen Content, and Nitrotyrosine,OPN and AP Expression in Aortic LesionsMonocyte macrophages infiltration, collagen content and ni-trotyrosine were measured as reported previously.10,11 Snapfrozen sections were analyzed by immunohistochemicalstaining for OPN expression. Polyclonal rabbit anti-mouseOPN antibody (Assay Designs, Ann Arbor, Michigan) wasadded in blocking buffer (1% bovine serum albumin in PBS)in a 1:100 dilution and incubated at 4C overnight. Afterrinsing with PBS biotinylated goat anti-rabbit antibody(Vector Laboratories, Burlingame, California) was added inblocking buffer in a dilution of 1:300 and incubated for 30

FIG. 1. Serum total cholesterol at sacrifice in 4 study groups. Effectof CKD on total cholesterol in 6 to 8 preparations per group ANOVAp �0.0001. Effect of simvastatin on total cholesterol in 6 to 8preparations per group ANOVA p � 0.5. Interaction in 6 to 8preparations per group ANOVA p � 0.3.

FIG. 2. Atherosclerotic lesion area in thoracic aorta in apoE�/� mic

6 to 8 preparations per group ANOVA p �0.01. Effect of simvastatin on ap � 0.8. Interaction in 6 to 8 preparations per group ANOVA p � 0.9.

minutes at room temperature. Peroxidase avidin-biotin com-plex reagent and AEC (3-amino-9-ethyl-carbazole) chromo-genic substrate were applied according to a commercial pro-tocol (Vector Laboratories).

Snap frozen sections were analyzed using the commercialworking solution used for detecting AP activity conjugatedantibodies (Vector Laboratories) according to manufacturerinstructions. OPN and AP expression was measured insideand outside the lesions, reflecting atherosclerotic and medialstaining, respectively.

Blood Pressure MeasurementsWe measured mean arterial blood pressure at the day ofsacrifice using direct intra-arterial recording by a previouslyestablished method.10

Statistical AnalysisData were analyzed by ANOVA, and the chi-square andStudent unpaired t tests, as appropriate. Results are ex-pressed as the mean � SEM. Differences between groupswere considered significant at p �0.05.

RESULTS

Effects of Simvastatin on SerumBiochemistry and Renal HistologyBody weight was comparable in the 4 study groups (table 1).After surgical creation of CKD the serum urea concentra-tion was increased 200% above baseline values. It de-creased in response to statin treatment in nonCKD andCKD mice (p � 0.04 and 0.006), whereas it increased inplacebo treated nonCKD mice and remained unchanged inplacebo treated CKD mice (p �0.05 and NS, respectively,table 1). Light microscopy examination of the remnant kid-ney tissue revealed severe cortical atrophy, increased inter-stitial fibrosis and enlargement of the glomerular surface,probably due to the hyperfiltration associated with CKD.There were no visible effects of statin treatment on thesechanges, probably due to the severity of the surgically cre-ated lesions or to the delay of statin introduction late afterlesion initiation by electrocautery.

h or without CKD. Effect of CKD on atherosclerosis progression in

e wit therosclerotic progression in 6 to 8 preparations per group ANOVA

NOVprepa

SIMVASTATIN IN APOLIPOPROTEIN E DEFICIENT MICE WITH RENAL FAILURE1634

Before the start of treatment CKD mice showed severehypercholesterolemia (data not shown). However, 4 weeks ofsimvastatin treatment did not significantly alter serum cho-lesterol levels in CKD or control mice (fig. 1).

CKD animals had a significantly higher serum calciumconcentration than nonCKD animals (p �0.001, table 1).Hypercalcemia was not modified by simvastatin treatment.The phosphorus concentration was comparable in the 4groups (table 1). The ratio of urinary protein/creatinine ex-cretion and mean arterial pressure did not differ among thegroups (table 1).

Atherosclerosis LesionsCKD mice had severe atherosclerotic lesions in the thoracicaorta that were significantly increased compared to those innonCKD controls (fig. 2). Long-term simvastatin treatmentdid not decrease uremia associated atherosclerosis in theaortic root or in the thoracic aorta. The same was true incontrol nonCKD mice (fig. 2).

Vascular CalcificationCKD animals showed a significant increase in vascular cal-cification at the intimal and medial aortic sites (fig. 3).Simvastatin treatment was associated with significantlylower plaque calcification in CKD mice than in CKD controls(fig. 3, A). Figure 4 shows calcium deposits in characteristicfeatures of CKD mouse aorta with and without simvastatintreatment. In contrast, simvastatin treatment had no sucheffect on medial calcification in CKD mice (fig. 3, B), nor didit modify the degree of intimal or medial vascular calcifica-tion in nonCKD control mice (fig. 3).

Total Collagen Content, Nitrotyrosineand Macrophage Infiltration in Aortic LesionsUremia was associated with a marked increase in plaquecollagen content. This increase was not significantly altered

FIG. 3. A, proportion of intima calcification area to total surface areaCKD. Effect of CKD on calcification progression in 6 to 8 preparatiprogression in 6 to 8 preparations per group ANOVA p � 0.3. Interof media calcified area to total surface area outside of atherosclerotCKD on calcification progression in 6 to 8 preparations per group Ato 8 preparations per group ANOVA p � 0.2. Interaction in 6 to 8

by simvastatin treatment (table 2). In contrast, simvastatin

treatment led to a significant decrease in nitrotyrosine ex-pression in the atheromatous plaques of CKD mice (Fisher’sexact test p �0.02, table 2). As revealed by monocyte-mac-rophage-2 staining the percent of lesion cross-sectional areaoccupied by macrophages, was comparable in the 4 groups(table 2).

OPN and AP ExpressionOPN and AP expression was measured inside and outside ofthe lesion, reflecting atherosclerotic and medial staining,respectively. OPN expression was significantly increased atthe 2 sites of the aorta in CKD mice compared that in tononCKD controls, whereas AP expression was significantlyincreased only at medial sites (table 2). Figure 5 shows thecharacteristic features of AP and OPN staining in aortictissues of a CKD mouse. Simvastatin had no effect on theexpression of either marker, although OPN expression wasnumerically higher in the lesions in simvastatin treatedCKD mice compared to that in placebo treated CKD mice(table 2).

therosclerotic lesions in aortic root of apoE�/� mice with or withouter group ANOVA p � 0.0006. Effect of simvastatin on calcificationn in 6 to 8 preparations per group ANOVA p �0.03. B, proportionions in aortic root of apoE�/� mice with or without CKD. Effect ofA p �0.0002. Effect of simvastatin on calcification progression in 6rations per group ANOVA p �0.3.

FIG. 4. Representative images. A, vascular calcification in controlapoE�/� mice with CKD. B, vascular calcification regression inapoE�/� mice with CKD treated with simvastatin. Black areas

of aons pactioic les

indicate calcification M, media. P, plaque. L, lumen. Eosin (2%) andvon Kossa silver nitrate staining, reduced from �25.

SIMVASTATIN IN APOLIPOPROTEIN E DEFICIENT MICE WITH RENAL FAILURE 1635

DISCUSSION

We evaluated the effects of HMG-CoA reductase inhibitor onuremia enhanced atherosclerosis and vascular calcificationin apoE�/� mice with superimposed CKD. We found thatCKD enhanced aortic plaque development in apoE�/� micecompared with controls, in agreement with previous reportsby us and others.10,12 Simvastatin treatment did not reducethe atherosclerotic lesions in the aortic root or thoracic aortain this experimental model, in line with its failure to de-crease serum total cholesterol. Nevertheless, simvastatintherapy led to a significant decrease in intima calcium con-tent, that is the calcium content of atheromatous plaques,although it did not change medial calcium deposition.

Vascular calcification is a prominent feature of CKD13

and it is predictive of increased cardiovascular morbidityand mortality.13 Coronary artery plaques in patients withend stage renal failure are characterized by increased me-dial thickness and marked intimal and medial calcification.3

Treatment with statins has been shown to be associatedwith a decrease in vascular calcification in 2 retrospectiveclinical studies4,14 and in animal models such as the apoELeiden mouse15 but not in another prospective trial.5 Thiseffect is generally associated with a decrease in serum lipidlevels, which was not the case in the current study.

The beneficial effect of simvastatin on CKD enhancedvascular calcification may also be explained at least in partby statin actions other than lipid lowering, including a re-

TABLE 2. Effect of simvastatin on nitrotyrosine expression, macrolesions of nonCKD a

Mean � SEMControl Placebo

Mean � SControl Simv

Nitrotyrosine expression (% mice) 0 33Macrophage staining (% lesions) 11.4 � 4.5 7.6 � 3Collagen expression (% lesions) 17.8 � 3.4 22.3 � 4% OPN:

Inside 2.8 � 1.4 3.4 � 1Outside 0.06 � 0.05 0.005 � 0

% AP:Inside 5.7 � 2.8 2.3 � 2Outside 1.8 � 0.5 2.3 � 0

Data were analyzed with ANOVA with 2 factors (CKD state and simvastatintest for global evaluation or ANOVA with 2 factors (CKD state/simvastatin* Vs control p � 0.02.† Vs CKD placebo p � 0.02.

�/�

FIG. 5. Representative images of vascular calcification in apoE micestaining (arrow). Reduced from �25.

duction of oxidative stress. Patients with uremia have dys-regulation of the immune system and they show excessivegeneration of reactive oxygen species. In the current settingsimvastatin treatment was associated with a decrease inlocal nitrotyrosine expression in the aorta of CKD mice,supporting the hypothesis of a link between oxidative stressand vascular calcification in the uremic state. Although lipidlowering by itself can decrease vascular oxidative stress,some of the antioxidant effects of statins appear to be cho-lesterol independent. For example, statins attenuate angio-tensin II induced free radical production in vascular smoothmuscle cells by inhibiting Rac1 mediated nicotinamide ade-nine dinucleotide oxidase activity and down-regulating an-giotensin-1 receptor expression.16 Moreover, statin therapydecreased serum nitrotyrosine levels significantly and inde-pendently of alterations in lipoproteins and inflammatorymarkers such as C reactive protein in patients with coronaryartery disease.17

An alternative explanation for the observed decrease inplaque calcification by simvastatin in this model could be aneffect on immediate calcification regulatory factors and pro-cesses. AP is a first candidate. It is a functional phenotypicmarker of osteoblasts and its activity is often used as amolecular marker for vascular calcification. It also is anearly marker of extracellular matrix deposition.18 OPN is asecond candidate. It is considered an inhibitor of calcifica-tion.19 In the current study we observed an increase in AP

e infiltration, collagen content, OPN and AP expression in aorticKD apoE�/� mice

nMean � SEMCKD Placebo

Mean � SEMCKD Simvastatin

CKD/Statin/InteractionEffect p Value

71* 11† �0.027.4 � 4.0 8.8 � 4.0 NS/NS/NS

32.2 � 5.3 26.0 � 1.9 �0.03/NS/NS

5.4 � 1.5 8.8 � 1.1 0.013/NS/NS0.56 � 0.27 0.50 � 0.13 0.017/NS/NS

6.1 � 2.9 7.8 � 1.9 NS/NS/NS3.7 � 1.3 5.3 � 1.1 0.060/NS/NS

idered but for nitrotyrosine expression data were analyzed by Fisher’s exactinteraction) considered.

phagnd C

EMastati

.2

.8

.0

.005

.2

.1

) consand

with CKD. A, von Kossa staining. B, AP staining (arrow). C, OPN

SIMVASTATIN IN APOLIPOPROTEIN E DEFICIENT MICE WITH RENAL FAILURE1636

expression outside atheromatous lesions and of OPN expres-sion inside and outside lesions in uremic mice compared tothose in controls. This goes along with the observed increasein vascular calcification in uremic mice. In line with thisobservation Moe et al recently reported the expression ofOPN, AP, type I collagen and bone sialoprotein in vascularsmooth muscle cells in the arteries of patients with uremiawho underwent renal transplantation.20 However, we failedto observe a significant effect of simvastatin on these pro-teins except for a trend of an increase of OPN inside thelesions in simvastatin treated CKD mice.

Possible direct effects of statins on vascular smooth mus-cle cells cannot be excluded. Recently it was reported thatstatins inhibit inorganic phosphate induced human aorticsmooth muscle cell calcification in vitro by preventing apo-ptosis via restoration of the growth arrest specific gene 6 andits receptor Axl.18,21 Regulation of the growth arrest specificgene 6 seems to occur at the posttranscriptional level as amevalonate pathway independent effect.21 Whether the anti-apoptotic effects of statins are related to their antioxidanteffects and whether these 2 mechanisms participate inde-pendently or jointly in their calcification inhibitory actionremain to be evaluated.

CONCLUSIONS

The current findings show a beneficial effect of simvastatinon CKD enhanced vascular calcification in apoE�/� mice.This effect was observed in the absence of changes in serumtotal cholesterol and atherosclerosis progression. Our obser-vation opens the possibility of a cholesterol independentaction of statins on vascular calcification via effects on oxi-dative stress. Further studies are required to determinewhether similar statin effects can be observed in patientswith CKD.

Abbreviations and Acronyms

AP � alkaline phosphataseapoE�/� � apolipoprotein E deficient

CAC � coronary artery calcificationCKD � chronic kidney disease

NS � not significantOPN � osteopontinPBS � phosphate buffered saline

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