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
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
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
method and presented as relative expression (RQ value) of
transcripts normalized to
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, 5⬘3/⬘ 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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