Activated vitamin D attenuates left ventricular abnormalities induced by dietary sodium in Dahl salt-sensitive animals

ArticleinProceedings of the National Academy of Sciences 104(43):16810-5 · November 2007with15 Reads
Impact Factor: 9.67 · DOI: 10.1073/pnas.0611202104 · Source: PubMed
Abstract

Observations in hemodialysis patients suggest a survival advantage associated with activated vitamin D therapy. Left ventricular (LV) structural and functional abnormalities are strongly linked with hemodialysis mortality. Here, we investigated whether paricalcitol (PC, 19-nor-1,25(OH)(2)D(2)), an activated vitamin D compound, attenuates the development of LV abnormalities in the Dahl salt-sensitive (DSS) rat and whether humans demonstrate comparable findings. Compared with DSS rats fed a high-salt (HS) diet (6% NaCl for 6 weeks), HS+PC was associated with lower heart and lung weights, reduced LV mass, posterior wall thickness and end diastolic pressures, and increased fractional shortening. Blood pressures did not significantly differ between the HS groups. Plasma brain natriuretic peptide levels, and cardiac mRNA expression of brain natriuretic peptide, atrial natriuretic factor, and renin were significantly reduced in the HS+PC animals. Microarray analyses revealed 45 specific HS genes modified by PC. In a retrospective pilot study of hemodialysis patients, PC-treated subjects demonstrated improved diastolic function and a reduction in LV septal and posterior wall thickness by echocardiography compared with untreated patients. In summary, PC attenuates the development of LV alterations in DSS rats, and these effects should be examined in human clinical trials.

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Available from: Juan Carlos Ayus
Activated vitamin D attenuates left ventricular
abnormalities induced by dietary sodium
in Dahl salt-sensitive animals
Natalya Bodyak*, Juan Carlos Ayus
, Steven Achinger
, Venkatesha Shivalingappa
, Qingen Ke*, Yee-Shiuan Chen*,
Debra L. Rigor*, Isaac Stillman
, Hector Tamez
§
, Paul E. Kroeger
, Ruth R. Wu-Wong
, S. Ananth Karumanchi
,
Ravi Thadhani
§
, and Peter M. Kang*
*Cardiovascular Division and
Division of Nephrology, Beth Israel Deaconess Medical Center, Boston, MA 02215;
Division of Nephrology, University
of Texas Health Science Center, San Antonio, TX 78229;
Abbott Laboratories, Abbott Park, IL 60064; and
§
Renal Unit and the Center for D-receptor
Activation Research (CeDAR), Massachusetts General Hospital, Boston, MA 02114
Edited by John T. Potts, Jr., Massachusetts General Hospital, Charlestown, MA, and approved September 7, 2007 (received for review December 15, 2006)
Observations in hemodialysis patients suggest a survival advan-
tage associated with activated vitamin D therapy. Left ventricular
(LV) structural and functional abnormalities are strongly linked
with hemodialysis mortality. Here, we investigated whether pari-
calcitol (PC, 19-nor-1,25(OH)
2
D
2
), an activated vitamin D com-
pound, attenuates the development of LV abnormalities in the
Dahl salt-sensitive (DSS) rat and whether humans demonstrate
comparable findings. Compared with DSS rats fed a high-salt (HS)
diet (6% NaCl for 6 weeks), HSPC was associated with lower heart
and lung weights, reduced LV mass, posterior wall thickness and
end diastolic pressures, and increased fractional shortening. Blood
pressures did not significantly differ between the HS groups.
Plasma brain natriuretic peptide levels, and cardiac mRNA expres-
sion of brain natriuretic peptide, atrial natriuretic factor, and renin
were significantly reduced in the HSPC animals. Microarray
analyses revealed 45 specific HS genes modified by PC. In a
retrospective pilot study of hemodialysis patients, PC-treated sub-
jects demonstrated improved diastolic function and a reduction in
LV septal and posterior wall thickness by echocardiography com-
pared with untreated patients. In summary, PC attenuates the
development of LV alterations in DSS rats, and these effects should
be examined in human clinical trials.
cardiac hypertrophy heart failure paricalcitol renal failure
T
he rate of cardiovascular-related mortality in hemodialysis
patients is 10–20 times higher than that observed in the
general population (1). Lef t ventricular hypertrophy (LVH) and
diastolic dysfunction are present in 50% of patients at dialysis
in itiation, and these abnormalities are strongly linked with
dialysis-related mortality (2). Currently, there are no well ac-
cepted means to modif y cardiac str uctural and functional alter-
ations in renal-failure patients.
We recently demonstrated in observational studies that ther-
apy with activated vitamin D to chronic hemodialysis patients is
associated with reduction in cardiovascular-related mortality (3,
4). Conversion of nutritional vitamin D (25(OH)D
3
)tothe
hor monally active form of vit amin D (1,25(OH)
2
D
3
) occurs
primarily in the kidney; thus, patients with kidney failure com-
monly present with altered vitamin D status (5). There is
growing evidence that vitamin D either directly or indirectly
af fects cardiac str ucture and function. The vitamin D receptor
k nockout mouse model demonstrates increased cardiac renin
ex pression and marked cardiomyoc yte hypertrophy (6), and
1,25(OH)
2
D
3
attenuates cardiomyocyte proliferation (7) and
hypertrophy (8) in vitro. Here, we demonstrate that treatment
with an activated vit amin D compound attenuates the develop-
ment of cardiac hypertrophy and dysfunction in a recognized
an imal model of such abnormalities and that comparable find-
ings are evident in humans.
Results
Effect of Paricalcitol (PC) on Cardiac Hypertrophy and Dysfunction. To
evaluate the effect of activated vitamin D on cardiac str ucture
and function, we chose the previously characterized high-salt
(HS)-induced model of hypertension and LVH in which Dahl
salt-sensitive (DSS) rats develop LVH and signs of diastolic
dysfunction as early as 68 weeks after HS exposure (9, 10).
Organ weights were adjusted to the tibial length (Table 1). Rats
receiving HSvehicle (V) for 6 weeks developed a significant
increase in heart and lung weights compared with their normal-
diet littermates (Table 1). In HS an imals, PC treatment resulted
in a 13% and 30% reduction in nor malized heart and lung
weights, respectively, compared with HSV-treated an imals.
Specific examination of individual cardiomyocyte size showed a
71% increase after HSV that was decreased by 21% in the
HSPC model [supporting information (SI) Fig. 5A and SI
Text]. Overall myocardial fibrosis was mild and did not differ
bet ween groups (SI Fig. 5 B and C).
We next examined cardiac parameters using M-mode echo-
cardiogram measures. In HSV animals, anterior and posterior
wall thickness was increased in the setting of reduced fractional
shorten ing (FS) (Table 2). In contrast, alternations in both wall
thick ness and FS were markedly attenuated in the HSPC group
(Table 2 and Fig. 1A). In addition, LV mass was elevated in the
HSV group, and sign ificantly reduced by PC treatment (Table
2). Thus, PC resulted in significant attenuation of cardiac and
cardiomyocyte hypertrophy and cardiac dysfunction.
Effect of PC on LV Hemodynamics and Blood Pressure. LV end-
diastolic pressure (LVEDP), a measure of LV filling pressure
reflecting LV dysfunction, was significantly elevated in the
HSV-treated group (Fig. 1 B and C). In contrast, HSPC
Author contributions: N.B. and J.C.A. contributed equally to this work; R.T. and P.M.K.
contributed equally to this work; N.B., J.C.A., S.A.K., R.T., and P.M.K. designed research;
N.B., J.C.A., S.A., V.S., Q.K., Y.-S.C., D.L.R., I.S., H.T., R.R.W.-W., S.A.K., R.T., and P.M.K.
performed research; N.B., J.C.A., I.S., P.E.K., S.A.K., R.T., and P.M.K. contributed new
reagents/analytic tools; N.B., J.C.A., S.A., I.S., P.E.K., R.R.W.-W., S.A.K., R.T., and P.M.K.
analyzed data; and N.B., J.C.A., I.S., P.E.K., S.A.K., R.T., and P.M.K. wrote the paper.
Conflict of interest statement: The study was supported in part by Abbott Laboratories.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
Abbreviations: ANF, atrial natriuretic factor; BNP, brain natriuretic peptide; DSS, Dahl
salt-sensitive; HS, high salt; LV, left ventricular; LVEDP; LV end-diastolic pressure; LVH, LV
hypertrophy; MAP, mean arterial pressure; PC, paricalcitol; V, vehicle; VDR, vitamin D
receptor.
To whom correspondence should be addressed at: Bullfinch 127, Massachusetts General
Hospital, Boston, MA 02114. E-mail: thadhani.ravi@mgh.harvard.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0611202104/DC1.
© 2007 by The National Academy of Sciences of the USA
16810–16815
PNAS
October 23, 2007
vol. 104
no. 43 www.pnas.orgcgidoi10.1073pnas.0611202104
Page 1
an imals had nearly normal LVEDP measures, suggesting that PC
prevented LV hemodynamic alterations in the setting of HS. We
then ascertained whether PC exerted its effect through changes
in blood pressure. In both HSV and HSPC animals, tail-
cuf f-measured mean arterial pressures (MAPs) increased com-
pared with controls. PC did not significantly reduce MAPs in
either the HS or normal-diet animals (Fig. 1D). To more
precisely exclude the effect of blood pressure explaining our
results, we used continuous wireless telemetry for seven con-
secutive days in nonanesthetized animals fed HS diet for 5 weeks
and injected with PC or V (SI Fig. 6 and SI Text). Blood pressures
were similar, providing further evidence against the possibility
that PC effects were mediated by changes in blood pressure.
Biochemical and Molecular Measures of Cardiac Dysfunction Attenu-
ated by PC.
Brain natriuretic peptide (BNP) and atrial natriuretic
factor (ANF) are reliable early measures of ventricular myocyte
stress and antedate cardiac hypertrophy and heart failure. HSV
an imals demonstrated a marked late increase in plasma BNP
levels compared with HSPC animals (Fig. 2A). Similarly,
cardiac ventricle mRNA expression of ANF and BNP was
increased in the HSV group and markedly attenuated in the
HSPC animals (Fig. 2 B and C). Because the v itamin D
receptor (VDR)KO model demonstrates cardiac myocyte hy-
pertrophy and an increase in cardiac renin gene expression (6),
and local renin-angiotensin activation is directly linked with
cardiac hypertrophy and heart failure (11), we also characterized
ren in mRNA expression in ventricular tissue. Overall, renin
ex pression in controls was low as suggested by previous studies
(12). In contrast, renin ex pression by RT-PCR was significantly
increased in the HSV group and markedly attenuated in the
HSPC model (Fig. 2 D and E). Real-time qPCR for ren in
mRNA ex pression c onfirmed these findings: HSV, 11 3-fold
increase compared with controls; HSPC significantly repressed
this alteration to 5 1-fold increase compared with controls;
HSV vs. HSPC, P 0.05. Thus, PC significantly attenuated
the biochemical and molecular measures of cardiac ventricular
stress and hypertrophy.
Ser um creatinine (HS V, 1.7 0.2 vs. HSPC, 1.8 0.2
mg/dl; P 0.05) and calcium (13.6 2.1 vs. 15.3 2.0 mg/dl,
respectively; P 0.05) levels did not significantly differ between
the two HS groups. Finally, after HS diet, the DSS model is
k nown to develop 25(OH)D
3
deficienc y (13), a finding we
verified after 6 weeks of HS (SI Fig. 7).
Effect of PC on Gene Expression Profiles. In an effort to examine the
potential mediators of PC’s ef fect, we first compared the cardiac
ventricle gene expression profiles of nor mal dietV and HSV
an imals. Among the 15,866 sequences evaluated, 1,338 genes
meeting a significance of P 0.001 were altered by HS: 741
(4.7%) down- and 597 (3.8%) up-regulated (Fig. 3A). By com-
parison, in the normal dietV compared w ith the HSPC
group, only 290 (1.8%) sequences were significantly down- and
231 (1.5%) up-regulated. Clustering the two data sets (normal vs.
HSV and normal vs. HSPC) suggested indirectly that PC had
a ‘‘normalizing’’ effect on several HS-induced genes (Fig. 3B and
SI Table 3). We examined cardiac microarrays from animals
af ter 3 weeks of HS to better elucidate the potential mechanisms
af fected by PC. Less than 5% of all expression changes observed
at 6 weeks in the HSV an imals were altered at 3 weeks, and
none met a significance of P 0.001. Nevertheless, gene
ex pression of muscle contractile proteins did demonstrate early
alterations (SI Table 4).
To more directly examine PC’s effect, we then focused on the
1,338 HS sequences that retained a P 0.001 when comparing
gene expression differences between HSV and HSPC groups
(Fig. 3C). This analysis yielded 45 specific HS sequences that
were significantly modulated by PC (SI Table 5). Among these,
Table 1. Morphometry analysis of DSS rats treated with PC
Normal diet HS diet
CPC C PC
Heart weight, g 1.3 0.02 1.2 0.01 1.7 0.02* 1.4 0.05
Lung weight, g 1.7 0.11 1.4 0.03 2.2 0.13 1.5 0.06
Liver weight, g 14.0 0.6 13.1 0.2 16.1 0.2 14.8 0.7
Tibial length, mm 38 0.2 37 0.2 37 0.3 37 0.2
HW/TL, mg/mm 34.5 0.5 32.3 0.1 44.8 0.4* 39.1 1.2*
LuW/TL, mg/mm 44.3 3.1 36.76 0.8 58.8 3.4* 41.1 1.6
LiW/TL, mg/mm 364 13 352 4 436 5* 406 19*
Results are presented as mean SEM.
*
, P 0.05 vs. normal diet treated with V. †, P 0.05 vs. HS diet treated
with V. C, V control; HW, heart weight; LuW, lung weight; LiW, liver weight; TL, tibial length.
Table 2. Echocardiographic analysis of DSS rats treated with PC
Normal diet HS diet
CPC C PC
HR, beats per min 394 13 405 8 352 22* 360 8
AWd, mm 1.72 0.07 1.65 0.06 2.14 0.04* 1.91 0.05*
PWd, mm 1.82 0.06 1.81 0.07 2.37 0.10* 1.88 0.04
LVDd, mm 7.82 0.18 7.73 0.22 7.80 0.27 8.35 0.05
LVDs, mm 3.70 0.14 3.73 0.11 4.25 0.22 3.93 0.08
LV mass, g 0.83 0.03 0.79 0.02 1.17 0.05* 1.01 0.02*
FS, % 52.7 1.1 51.6 0.8 45.7 1.6* 52.9 1.0
Results are presented as mean SEM.
*
, P 0.05 vs. normal diet treated with V. †, P 0.05 vs. HS diet treated
with V. C, V control; HR, heart rate; AWd, anterior wall thickness in diastole; PWd, posterior wall thickness in
diastole; LVDd, LV dimension in diastole; LVDs, LV dimension in systole, LV mass, calculated LV mass; FS, fractional
shortening.
Bodyak et al. PNAS
October 23, 2007
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APPLIED BIOLOGICAL
SCIENCES
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possible candidates for PC’s ef fect included genes related to cell
signaling (Nkiras2), cell adhesion (Itgb1, Nrp1), stress responses
(Hmox1), and muscle contractile function (Myh7, Tpm1). Im-
port antly, myosin heavy-chain (MHC) isofor m switch (increased
-MHC w ith decreased
-MHC expression), an established
fet al-type gene reexpression characteristic of load-dependent
hypertrophy (14), was reversed after PC treatment.
PC Is Associated with Improved LV Parameters in Hemodialysis Pa-
tients.
In the U.S., PC is routinely used among chronic hemo-
dialysis patients (3), a population with a high prevalence of
cardiac hypertrophy, diastolic dysfunction, and profound vitamin
D deficiency similar to our animal model (15). Therefore, we
retrospectively examined baseline and 12-month cardiac echo-
cardiograms in adult hemodialysis patients treated with or
without PC (SI Table 6 for baseline characteristics). Among
patients untreated for the entire duration, E/A ratios decreased,
suggesting a worsening of diastolic dysfunction, whereas, in
treated patients (duration of PC, 4.3 1.2 months; average dose,
13 7
g/week), E/A ratios increased (Fig. 4A). Furthermore,
treatment was associated with a 15% and 11% reduction in
sept al and posterior wall thickness, respectively (Fig. 4 B and C).
Ejection fractions were within the normal range at baseline and
follow-up (Fig. 4D). Although observational and retrospective,
these findings suggest that our animal results may translate to
humans.
Discussion
We have shown that in the DSS model, PC markedly attenuates
the development of the anatomic, functional, biochemical, mo-
lecular, and genetic alterations characteristic of cardiac hyper-
trophy and dysfunction. The DSS model has been reproducibly
used to investigate cardiac hypertrophy, diastolic dysfunction,
and transition to heart failure (9, 16). Therapies shown to
attenuate cardiac dysfunction in this model (17, 18) ameliorate
c omparable cardiac abnormalities in humans (19). Importantly,
although our retrospective clinical data of activated vit amin
D-treated hemodialysis subjects involved small numbers, the
observed significant improvement in cardiac alterations is en-
c ouraging and suggestive of a potential for clinical intervention.
These results, the accumulating data on the cardiovascular
ef fects of activated vitamin D, and studies from our group and
others suggesting a survival benefit associated with activated
vit amin D therapy in dialysis patients (3, 4, 20) strongly support
the basis for an interventional trial to directly assess the cardiac-
specific effects of activated vit amin D administration in renal-
failure subjects.
Sk in-derived 7-dehydrocholesterol undergoes liver hydroxyla-
tion to form 25(OH)D
3
, the storage form of vitamin D (21). The
k idney hydroxylates 25(OH)D
3
to 1,25(OH)
2
D
3
, the active form
of vitamin D that exerts its effect through binding the nuclear
VDR present in cells, including smooth muscle and endothelial
cells, and possibly cardiomyocytes (7, 22, 23). A 25(OH)D
3
deficienc y leads to derangements in cardiac myocyte contraction
(24), proliferation (25), and cardiac c ollagen accumulation (26).
Further more, 1,25(OH)
2
D
3
blocks endothelin-induced ventric-
ular myocy te hypertrophy (8), and VDRKO mice develop car-
diomyoc yte hypertrophy (6). Comparable human dat a has been
limited. Nutritional vitamin D-deficienc y rickets is associated
Fig. 2. Plasma BNP and LV gene expression. (AC) Plasma BNP level (ELISA)
(A), and relative ANF and BNP (B and C) mRNA level in LV tissue, respectively,
(real time RT-PCR). C, control vehicle group. n 4 –11 in each group.
*
, P
0.05. (D) Semiquantitative RT-PCR of LV renin mRNA expression. Actin expres-
sion is shown below for loading control. (E) Quantification of renin mRNA
expression normalized to actin mRNA expression.
*
, P 0.05.
Fig. 1. Echocardiographic and hemodynamic measures. (A) Representative
M-mode images of the LV of DSS rats fed normal and HS diets. The increases
in LV wall thickness and lower LV wall motion in HS animals are improved in
the HSPC group. AW, anterior wall; PW, posterior wall; LVC, left ventricular
cavity. (B) Representative LV pressure tracings. (C) LVEDP measurement.
LVEDP is significantly higher in HS group, whereas in HSPC animals, it is
similar to controls. (D) Noninvasive MAP measurements. MAP is significantly
elevated in HS diet groups, but not modified by PC treatment. n 3–10 in each
group.
*
, P 0.05.
16812
www.pnas.orgcgidoi10.1073pnas.0611202104 Bodyak et al.
Page 3
with a dilated cardiomyopathy (27). Patients with heart failure
have reduced circulating levels of 25(OH)D
3
(28), and small
unc ontrolled studies suggest that 1,25(OH)
2
D
3
therapy reduces
LV hypertrophy in hemodialysis patients (29). Nevertheless,
before this study, the link between animal and human studies had
been tentative.
We used a high threshold of statistical significance throughout
our microarray gene analyses to minimize false-positive findings
in the setting of multiple testing (30). In this context, a number
of genes altered by HS diet were normalized by PC including the
myosin heav y-chain isoform switch characteristic of load-
dependent hypertrophy (10, 14). Changes in
-tropomyosin and
in the NF-
B pathway, which have been linked with ventricular
hypertrophy (31, 32), were also positively modified by PC. This
is consistent with the finding that 1,25(OH)
2
D
3
sign ificantly
blunts the activation of NF-
B signaling in vitro (33). Finally,
cardiac renin gene expression was attenuated by PC, a finding
c onsistent with Li et al . (6, 34) who demonstrated that
1,25(OH)
2
D
3
is a negative regulator of renin gene synthesis.
Whether the repression of cardiac ren in by PC is cause or
c onsequence of LV improvement remains unknown. Evidence
for the presence of a functional VDR in cardiomyocytes is
lack ing, hence the effect of PC may be indirect. For example,
mast cells release renin and activate the local cardiac RAS
system (35), and mast cells c ontain the VDR (36). In the setting
of cardiac hypertrophy, however, the precise role of locally
derived renin c ompared with that released from the kidney
c ontinues to be debated (35, 37).
Import antly, DSS rats develop significant 25(OH)D
3
defi-
cienc y (13), as we confirmed, and we suggest that activated
vit amin D replacement significantly attenuates the LV alter-
ations characteristic of this model. Furthermore, our animal
findings may translate to humans with vitamin D deficienc y and
LV alterations such as those with kidney disease, although we
ack nowledge that the DSS model lacked the uremic milieu of
renal failure, and our human data were observational and
retrospective. Indeed, a more parallel comparison may be the
examination of PC’s effect in a uremic model of heart failure.
Nonetheless, PC is already in routine clinical use among patients
with renal disease, and thus an interventional clin ical trial would
be the next logical step. Although many unanswered questions
remain in the rapidly expanding area of the ‘‘cardio-renal
syndrome,’’ alterations in the vitamin D axis may be at least one
integral mediator of this highly prevalent condition.
Materials and Methods
Animal Model. Male DSS rats (Harlan Sprague–Dawley) were
bred and fed a nor mal diet to 6 weeks of age. To generate
pressure-overload cardiac hypertrophy, an imals were fed a HS
diet (6% NaCl) for the following 6 weeks. To study the effects
of PC, the animals were divided into four groups: (i) normal
dietV, (ii) normal dietPC (200 ng i.p. three times per wk),
(iii)HSV, and (iv)HS PC (200 ng i.p. three times per wk).
PC (19-nor-1,25-(OH)
2
D
2
), which is 10 times less potent in
gastrointestinal calcium and phosphor us absorption than the
endogenous form of active vitamin D calcitriol (1,25(OH)
2
D
3
),
was prepared with 95% propylene glycol/5% ethyl alcohol
solution and initiated i.p. 3 days before the initiation of HS diet
and continued at 200 ng three times per week (Monday, Wednes-
day, Friday) thereafter. Administration of 200 ng of PC three
times per wk was based on comparable studies of nephrecto-
mized uremic rats, where doses of up to 250 ng i.p. three times
per week were safely administered w ithout significant changes in
blood pressure or ser um calcium or phosphorus (38, 39). Ne-
Fig. 3. Summary of gene expression changes. (A) Normal diet vs. HSV
‘‘M/A’’ plot [average probe intensity (x axis) vs. log ratio (y axis)] using a P
0.001 as cut-off for significance. Specifically, 1,338 sequences are regulated
(597 up- and 741 down-regulated) by HS compared with normal diet at 6
weeks. Scale is log ratio 2.0 to 2.0 (i.e., 100- to 100-fold). (B) Agglom-
erative hierarchical clustering using the 1,338 HS sequences and two samples
(normal diet vs. HSV; normal diet vs. HSPC). Scale is log ratio 0.7 to 0.7
(i.e., 5- to 5-fold). Red, up-regulation and blue, down-regulation. A se-
quence that failed to meet the P value 0.001 cut-off was colored black. (C)
‘‘M/A’’ plot of the 6-week gene expression pattern for HSV vs. HSPC using
the 6-week 1,338 HS sequences (P 0.001). Of the 1,338 sequences regulated
at 6 weeks, only 45 are regulated specifically by PC (22 up- and 23
down-regulated).
Fig. 4. Quantitative analysis of echocardiogram parameters in hemodialysis
patients. Changes in E/A ratio (A), LV septal thickness (B), posterior wall (PW)
thickness (C), and ejection fraction (D) in patients with (n 15) and without
(n 6) PC treatment.
Bodyak et al. PNAS
October 23, 2007
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phrectomy was not part of this study. Controls were age-matched
male DSS rats fed normal chow receiving equal volume of the V
three times per week for 6 weeks starting at 6 weeks of age. Af ter
6 weeks of normal diet or HS, morphometric measures, cardiac
function using noninvasive and invasive physiologic methods,
histopathology, and biochemical/gene changes related to cardiac
stress were examined. The Institutional Animal Care and Use
Committee (IACUC) of the Beth Israel Deaconess Medical
Center approved this study.
Echocardiography. Echocardiogram was performed under isoflu-
rane anesthesia. An Agilent Sonos 5500 sector scanner equipped
with a 7.5-MHz phased-array transducer was used to rec ord
2D-guided M-mode tracings. The leading-edge method was used
to determine anterior and posterior wall thickness and LV
internal dimensions. LV mass was calculated by using the
c orrected American Society of Echocardiography (ASE) sim-
plified cubed equation: LV mass (g rams) 0.8 [1.05
(LVDdAWdPWd)
3
(LVDd)
3
].
Blood Pressure and LV Pressure Measurements. Noninvasive arterial
blood pressure was measured with an automated noninvasive
t ail-cuff BP-2000 Blood Pressure A nalysis System (Visitech
Systems, Apex, NC) as described (40). An average of 10 readings
per animal was assessed. Invasive LV pressure measurements
were obtained by inserting a 1.4 French microtip pressure
catheter (Millar Instruments, Houston, TX) through the right
c ommon carotid artery into the left ventricle. Data were re-
c orded by using a PowerL ab system and Chart 5 software (AD
Instr uments, Colorado Springs, CO). Heart rate, LV systolic
pressure, and LVEDP were measured directly from LV pressure
wavefor ms.
Plasma BNP. Tail-vein blood was collected at baseline, 3, and 6
weeks (at sacrifice) after initiation of a HS diet. Plasma levels of
BNP were measured by using BNP ELISA kit (Assaypro, St.
Charles, MO) according to the manufacturer’s instructions.
Real-Time PCR and RT-PCR Analyses. Tot al RNA was extracted from
rat ventricle tissue by using TRIzol (GIBCO–BRL, Gaithers-
burg, MD). RT-PCR was performed by PCR amplification of
cDNA reverse-transcribed from mRNA by using rat renin and
actin primers (SI Table 7). Real-time PCR primers for rat BNP,
ANF, renin, and actin were obtained from Applied Biosystems
(Foster City, CA) (SI Table 7). Real-time PCR was performed
with a 7500 Real-Time PCR System (Applied Biosystems). Each
sample had a final volume of 20
l, containing following: 1 one
step SYBR g reen master mix, 3–5 pmol of primers for the gene
of interest, and 2
g of RNA. Samples were run in duplicate in
optically clear 96-well plates. Data were calculated by 2
⫺⌬CT
method and presented as relative expression (RQ value) of
transcripts normalized to
-actin.
Histological Analysis. Cardiac histological analyses were per-
for med as described (9, 10). H&E- and Masson’s trichrome-
st ained sections were evaluated in a blinded fashion for patho-
logical changes. Measurements were made at 20 by using a
calibrated digit al camera and software (DP 70 and DPCon-
troller, Olympus, Irving, TX).
RNA Isolation for Microarray Analysis. Total RNA from left ven-
tricles was isolated by using Qiagen RNeasy procedures (Qiagen,
Valencia, CA). Hearts were removed rapidly, and the left
ventricles were cut and snap-frozen in liquid nitrogen. Total
RNA was isolated by homogenization in RLT buffer and
purification performed on the RNeasy Mini column. Quality of
rec overed RNA was assessed w ith the Agilent 2100 Bioanalyzer;
RIN scores were 9 for all samples.
Microarray Analysis. Generation of biotinylated cRNA probe for the
Affymetrix arrays was performed according to Affymetrix’s pro-
tocols. The Affymetrix Rat Genome 230A GeneChip (Affymetrix,
Santa Clara, CA) containing probe sets for 15,000 known se-
quences and expressed sequence tags (ESTs) was used for analysis.
Hybridization and scanning were done per standard Affymetrix
protocols. The re sulting raw data files (.cel file s) were first analyzed
with the R/Bioconductor AffyQC package, and no outliers were
observed with respect to percent present, 53/ ratios or all pair-wise
correlation analysis. Data were then loaded into the Rosetta
Resolver system, where it was normalized, replicates combined, and
ratios created by using the standard Affymetrix error model pipe-
line. Further analysis used the agglomerative clustering and data
visualization tools in Resolver. The Database for Annotation,
Visualization and Integrated Discovery (DAVID) (National Insti-
tute of Allergy and Infectious Diseases, Bethesda, MD) and Gen-
MAPP programs (University of California, San Francisco, CA)
were used for gene ontology analyses.
Human Studies. Adult chronic hemodialysis patients at the Texas
Diabetes Institute (San Antonio, TX) were enrolled in a 12-
month study to assess the ef fects of hemodialysis on cardiac
function (41). Baseline and 12-month transthoracic echocardio-
grams were performed by using standard techniques and re-
viewed by two independent cardiologists blinded to the exami-
nation date and therapy st atus. PC use was left up to the
discretion of the treating physician. An in itial cohort of 51
patients received thrice weekly conventional hemodialysis, and
among these, 38 underwent a baseline and 12-month echocar-
diographic evaluation. We included all who received either no
activated vitamin D therapy (n 6) or who received only PC for
at least one month (n 15). The study was approved by the IRB
of the University of Texas Health Science Center San Antonio,
and all participants provided written informed consent.
Statistical Analysis. Data were expressed as means SEM. Com-
parisons between and within groups were conducted with unpaired
Student t tests and repeated-measures ANOVA, respectively. P
values of 0.05 were considered statistically significant.
This work was supported in part by National Heart, Lung, and Blood
Institute Grants R01 HL65742 (to P.M.K.) and DK71674 (to R.T.) and
research support from Abbott (R.T.).
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