Download Different levels of aldosterone synthase affect the cardiovascular

R2 HYPERTENSION/2007/098897
SALT-SENSITIVE BLOOD PRESSURE IN MICE WITH INCREASED EXPRESSION
OF ALDOSTERONE SYNTHASE
Online Supplements (Makhanova et al.)
Online Expanded Materials and Methods
Blood pressure (BP) measurements
BPs were measured in conscious mice with a computerized tail-cuff system (Visitech Systems,
Cary, NC).
Blood analysis
Individual blood sample were taken for plasma measurements 4 days after completing of the
metabolic studies, except that aldosterone, angiotensin II, plasma renin activity were collected
during the terminal anesthesia with 2.5 % avertin, which was after about 6 weeks on LS or HS
diets. Plasma electrolyte concentrations were measured using a VT250 Chemical Analyzer
(Orthodiagnostic Clinical Inc.). Hematological analyses were performed with an Animal Blood
Counter (Heska Inc, CO). Plasma aldosterone was measured with a radioimmunoassay (RIA)
using the Coat-A-Count RIA procedure (Diagnostic Products, Los Angeles, CA). Plasma
corticosterone was determined with an RIA kit (ICN Biomedicals, Palo Alto, CA). Plasma renin
activity was measured as described previously by RIA (DiaSorin, Stillwater, Minnesota, USA)
(1). The concentration of ANG II was measured with an RIA kit (Peninsula Laboratories, San
Carlos, CA).
Echocardiographic assessment
Transthorasic echocardiography was performed using a Visual Sonics 660 Instrument and 30
MHz probe (Toronto, Canada) at the Cardiovascular Facility Core at the University of North
Carolina. A two-dimensional short-axis view of the left ventricle was obtained at the level of the
papillary muscles. M-mode measurements of heart rate, left ventricular end-diastolic dimension,
left ventricular end-systolic dimension, posterior wall thickness (end-systolic and end-diastolic)
and intraventricular wall septum thickness were made from the original tracings. The followings
variables were calculated: fractional shortening, left ventricular volume, left ventricular ejection
fraction, left ventricular mass, cardiac output.
Histological Analysis
Organs were fixed in 4% buffered paraformaldehyde overnight, embedded in paraffin, sectioned
and stained for light microscopy with hematoxylin and eosin, periodic acid-Schiff, or MassonTrichrome.
Real-Time RT-PCR
Expression of genes in the tissues was determined by quantitative real-time RT- PCR with an
Applied Biosystems 7700 Sequence Detection System (Perkin-Elmer), as described (2). Briefly,
whole kidneys, adrenal glands, and the apex of hearts were stored in "RNA later" solution
(Ambion, Austin, TX). Then, small pieces of tissue (50-100 mg) were added to lysis buffer
made by diluting 2× RNA lysis buffer (PE Biosystems, Foster City, CA) with Ca2+- and Mg2+free PBS. The tissues were macerated with a 1/4-inch cylindrical beads and tissue homogenates
were stored at
20°C for up to 16 h before use. RNA was isolated from tissue with the ABI
Prism 6700 automated nucleic acid workstation (PE Biosystems), following the manufacturer's
20°C. Real-time RT-PCR amplifications were
protocol. Total RNA samples were stored at
performed in a 96-well plate in the ABI Prism 7700 sequence detector (PE Biosystems) in a total
volume of 30 µl, which included 10 µl of RNA sample from the ABI Prism 6700 plus 20 µl of a
reaction mixture made with minor modifications of the manufacturer's instructions. Each RT-
2
PCR amplification was performed in duplicate. During the amplification, the fluorescence of
reporter dye was measured by the 7700 sequence detector in each well of the 96-well plate.
Relative levels of gene expression as a percentage of wild type were determined for each gene.
Analysis of kidney function
To determine 24 hour water and food intake, urine volume and excretion of electrolytes, mice
were housed in metabolic cages for 3 days. Urine osmolality was measured by freezing point
depression. Urine NOx was measured by a colorimetric assay ( Nitrate/Nitrite Colometric Assay
Kit, Cayman Chemical, Ann Arbor, MI).
Morphometric analysis
Image J software (NIH) was used to estimate the degree of fibrosis, defined as the ratio of Blue
Masson Trichrome stained area to the entire area. To evaluate the cross-sectional area of
cardiomyocytes, heart sections was stained with wheat germ agglutinin coupled with fluorescent
TRITC. The areas of myocytes that had been cut transversely in sections of the lateral mid-free
wall of the left ventricle were determined using Image J software and counting 100 cells per
animal.
Statistical analysis
All statistical analyses were with JMP statistical Software (SAS Institute, Cary, NC), and are
presented as means±SEM. Statistical significances were assessed with two-factor ANOVA. Post
hoc analyses were with the Student t-test.
3
Online References
1. Kim HS, Krege JH, Kluckman KD, Hagaman JR, Hodgin JB, Best CF, Jennette JC,
Coffman TM, Maeda N, Smithies O. Genetic control of blood pressure and the angiotensinogen
locus. Proc Natl Acad Sci U S A 1995;92:2735-2739.
2. Kim HS, Lee G, John SW, Maeda N, Smithies O. Molecular phenotyping for analyzing
subtle genetic effects in mice: application to an angiotensinogen gene titration. Proc Natl Acad
Sci U S A. 2002;99:4602-4607.
4
Figure legends
Figure S1
Plasma renin activity (PRA) and plasma ANG II in WT (white bars) and AShi/hi (black bars)
mice on LS, NS and HS diets: (A) PRA (n ≥7). (B) Plasma ANG II (n≥7). The data are
means±SEM.
Figure S2
Cardiac remodeling in AShi/hi and WTmice after ANG-II infusion: (A, B) Lower power images of
heart sections of WT (A) and AShi/hi (B) mice stained with wheat germ agglutinin coupled with
fluorescent TRITC. (C, D) Higher power images demonstrating an increase in perivascular
fibrosis in the AShi/hi mice (D) compared with WT mice (C). The data are means±SEM.
Figure S3
Cardiac fibrosis, cardiac Collagen I and Collagen III expression and urinary excretion of 8isoprostane in WT (white bar) and AShi/hi (black bar) mice fed a MHS diet following ANG II and
PBS infusion. (A) Cardiac fibrosis after PBS (n=3) and ANG II (n=5) infusion. (B) Cardiac
Collagen I mRNA level (relative to WT following PBS infusion as 100%) after PBS (n =7) and
ANG II (n=9) infusion. (C) Cardiac Collagen III mRNA level (relative to WT following PBS
infusion as 100%) after PBS (n =7) and ANG II (n=9) infusion. (D) Urinary excretion of 8isoprostane during PBS (n ≥3) and ANG II (n=5) infusion. Data are presented as means±SEM.
5
Tables
Table S1
Aldosterone and corticosterone in AShi/hi and WT mice on NS diet
Parameters
WT
AShi/hi
Adrenal AS mRNA (% WT)
100±17 (12)
150±17 (18)*
Adrenal aldosterone/ protein (ng/mg)
0.73±0.09 (9)
0.97±0.09 (11)
Plasma aldosterone (pg/ml)
576±102 (10)
700±160 (11)
Plasma corticosterone (ng/dl)
68±16 (8)
95±17 (8)
Values are means ± SE and parentheses are number of animals.* p<0.05 versus AS+/+ mice.
6
Table S2
Dietary salt and plasma electrolytes, urinary excretion of electrolytes and NOx (NO2 and NO3) in
AShi/hi and WT mice
Electrolytes
AShi/hi
WT
Plasma
Na+, mmol/L
LS
145.5±1.6 (6)
148.8±1.2 (9)
NS
143.08±0.31 (6)
143±0.7 (5)
HS
143.8± 1.7(10)
142.6±1.6 (11)
LS
5.6±0.2 (8)
5.5±0.1 (9)
NS
5.5±0.3 (6)
5.4±0.1 (9)
HS
5.4 ± 0.1(8)
5.0±0.1 (6)*
<5
<5
NS
68±17 (6)
90±35 (5)
HS
3124± 491 (6)
2832±448 (6)
UkV, μmol/day LS
377 ± 41 (6)
297 ± 37 (6)
NS
397 ± 54 (5)
347 ± 111 (5)
HS
500±71 (6)
438±54 (6)
NOx, μmol/day LS
568±118 (6)
601± 114 (6)
NS
882±216 (5)
520±206 (5)
HS
1169 ±119 (6) †
1155±92 (6) †
K+ , mmol/L
Urine
UNaV, μmol/day LS
Values are means ± SE and parentheses are number of animals. *P<0.05 versus WT on HS diet,
†P<0.01 versus same genotype on LS diet.
7
Table S3 Dietary salt and kidney gene expression in AShi/hi and WT mice
Gene
α-ENaC
NCC
NHE3
NKCC2
AQP2
eNOS
AShi/hi
WT
LS
144±18 (7)*
114± 10 (9)
NS
100±9 (8)
107±7 (7)
HS
61±3 (8) §
77±4 (8) † ‡
LS
96±8 (7)
85±5 (9)
NS
100±8 (8)
115±9 (7)
HS
90±10 (8)
94±7 (8)
LS
107±12 (12)
80±11 (9)*
NS
100±8 (8)
120±16 (7)
HS
108±9 (8)
128±12 (8)
LS
65±7 (7) †
67±12 (9) †
NS
100±7 (8)
134±16 (7)
HS
78±9 (8)
73±7 (8) †
LS
50± 7(7) †
80±11 (9)
NS
100±13 (8)
121±17 (7)
HS
123±14 (8)
124±13 (8)
LS
118±9(7)
90±3 (9)‡
NS
100±7 (8)
119±10 (7)
HS
113±16 (8)
122±12 (8)
Values are means ± SE and parentheses are number of animals. *P<0.05 versus same genotype
on NS diet, †P<0.01 versus same genotype on NS diet, ‡P<0.01 versus WT on same type of diet;
§P<0.001 versus same genotype on NS diet
8
Figure S1
9
Figure S2
10
Figure S3
11