2-1. Animals
Male Wistar rats (8 weeks old) with body weight in the range of 180-200 g (at age
of 7-8 weeks) were used for the study. All experiments were carried out with approval
of the Animal Care and Use Committee of the National Taiwan University and in
accordance with the Guide for the Care and Use of Laboratory Animals. The rats were
individually housed in plastic cages, maintained under controlled temperature at 24℃±1
℃ with a light-dark cycle of 12 hours each and free access to food and water
throughout the experimental period. For the part I study investigating the effectiveness
of OLM, the rats were allocated randomly to 3 groups of 12-15 each for comparison: (1)
normal control (NC), (2) chronic renal failure (CRF) and (3) OLM-treated CRF rats
(OLM+CRF). For the part II study investigating the effectiveness of PM, the rats were
allocated randomly to 4 groups of 12-15 each for comparison: (1) normal control (NC),
(2) chronic renal failure (CRF), (3) PM-treated normal controls (NC+PM) and (4)
PM-treated CRF rats (PM+CRF).
2-2. 5/6 subtotal nephrectomy (SNx) for CRF model
CRF was induced by 5/6 subtotal nephrectomy, which is the classic animal model.
Surgery for inducing CRF was performed under total anesthesia by intraperitoneal
injection of sodium pentobarbital (50 mg/kg) as previously described (Lin, Chen et al.
2002). After the right kidney was removed, two branches of the left renal artery were
ligated to create an infraction. Sham surgery was conducted in rats of the control group.
After recovering for one week, CRF rats received daily oral gavage with OLM (10
mg/kg/day) or placebo for the next 8 weeks in the first study. In the second study,
animals received daily intraperitoneal injection with PM (60 mg/Kg/day) or placebo
after one week recovery from the surgical procedure.
2-3. Assessment of hemodynamics
Blood pressure was measured by catheterization method in anesthetized animals 8
weeks after operations. The pulsatile aortic pressure was measured using a Millar
telemetric catheter with a high-fidelity pressure senor (mode CPS 320; size 2F; Millar
Instrument, Huston, TX) and an electromagnetic flow probe (model 100 series, internal
circumference 8 mm; Carolina Medical Electronics, King, NC). Mechanical ventilation
with a tidal volume of 5–6 ml/kg and respiratory rate of 50–70 breaths/min was
provided during the rats receiving tracheotomy. The catheter was introduced via the
isolated right carotid artery into the ascending aorta after the animals were anesthetized.
The chest was opened through the right second intercostal space. Pulsatile aortic flow
measurments were taken in the ascending aorta with an electromagnetic flowmeter
carefully positioned to give a clear pulse tracing. The electrocardiograph (ECG) of lead
II was recorded with a Gould ECG/Biotach amplifier (Gould Electronics, Cleveland,
OH). The analog waveforms were sampled at 500Hz using a 12-bit simultaneously
sampling analog-to digital converter (Acqutek Co., Taipei, Taiwan) interfaced to a
personal computer. Selection of signals of 5-10 beats at steady state was made on the
basis of the following criteria: recorded beats with optimal velocity profile that was
characterized by a steady diastolic level, maximal systolic amplitude and minimal late
systolic negative flow. The selective beats were averaged in the time domain using the
peak R wave of ECG signal as fiducial points. Timing between the pressure and flow
signals, because of the spatial distance between the flow probe and the proximal aortic
pressure transducer, was corrected by a time-domain approach, in which the foot of the
pressure waveform was realigned with that of the flow (Mitchell, Pfeffer et al. 1994).
The resulting pressure and flow signals were subjected to further vascular impedance
analysis.
2-4. Aortic input impedance spectra
The aortic input impedance was obtained from the ratio of the ascending aortic
pressure harmonics to the corresponding flow harmonics, using a standard Fourier
series expansion technique (Milnor 1989; Nichols and O'Rourke 1998; Chang, Hsu et al.
2003). Total peripheral resistance of the systemic circulation was calculated as the
mean aortic pressure divided by mean aortic flow rate. The aortic characteristic
impedance was computed by averaging high-frequency moduli of the aortic
input-impedance data points (4th–10th harmonics) (Huijberts, Wolffenbuttel et al. 1993;
Gaballa, Raya et al. 1999). Taking aortic characteristic impedance into consideration,
we calculated the systemic arterial compliance (C) at the mean aortic pressure (Pm) by
expanding the two-element Windkessel model into a three-element model (Liu, Brin et
al. 1986), which accounted for the nonlinear exponential pressure-volume relationship:
C(Pm) ＝
SV is the stroke volume, K is the ratio of total area under the aortic pressure curve to the
diastolic area (Ad), Zc is the aortic characteristic impedance, b is the coefficient in the
pressure-volume relationship (-0.0131 ± 0.009 in the aortic arch), Pi is the pressure at
the time of incisura, and Pd is the end-diastolic pressure.
The wave transit time can be computed by the impulse response of the filtered
aortic input impedance. This was accomplished by the inverse transformation of aortic
input impedance after multiplication of the first 12 harmonics by a Dolph-Chebychev
weighting function of the 24th order (Laxminarayan, Sipkema et al. 1978). Then, the
time-domain reflection factor was derived as the amplitude ratio of the
backward-to-forward peak pressure wave by the method proposed by Westerhof et al
(Westerhof, Sipkema et al. 1972). Therefore, both the wave transit time and the wave
reflection factor characterize the wave reflection phenomenon in the vasculature.
2-5. Sample preparations and renal function measurement
Body weight was measured weekly after the surgery. Eight weeks after OLM or
PM administration, blood, tissues samples of aorta and heart were collected under
anesthesia from 12 rats of each group. Blood samples were collected directly via cardiac
puncture and serum was obtained after centrifugation at 2000 g for 15 min at 4 ℃. All
samples were immediately refrigerated until analysis. Rats were then perfused with iced
phosphate buffered saline thoroughly and sacrificed to collect aorta and heart samples.
After weighted, the samples were cut into pieces and fixed in formalin or stored at －80
℃. Serum levels of creatinine and blood urea nitrogen (BUN) were measured to test
renal function by using an autoanalyzer system (Hitachi Model 7070, Hitachi
Electronics Co., Ltd., Tokyo, Japan).
2-6. Immunohistochemical analysis
Aorta sections (4m) were processed for immunochemistry as described
previously (Pathak, Gupta et al. 2008). Sections were deparaffinized in xylene and
hydrated through a series graded alcohol (100%, 90%, 70% and 50%) to distilled water,
followed by treatment with 3% H2O2/methanol and then blocked in 3% normal horse
serum for 20 min at room temperature. Anti-AGE MAb, clone 6D12 (1:50, Trans
Genetic Inc., Kumamoto, Japan) was used to incubate with the sections for 30 min at
room temperature. After washing 3 times with PBS, immunostaining was performed
using biotinylated anti-mouse secondary antibody (1:500, R.T.U. Vectastain Universal
Elite ABC kit, Vector Laboratories Inc., Burglingame, CA) and then avidin biotin
peroxidase complex (Vectastain ABC kit). The sections were then visualized by color reaction with diaminobenzidine (Vector Laboratories) and counterstained with hematoxylin.
2-7. Western blot analysis
The method used for analyzing collagen glycation in aortic well was applied in this
study (Turk, Misur et al. 1999). After aortic samples were extensively treated with
pepsin, proteinase K and collagenase, the tissue extracts with each of 40 g/l were
fractionated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) gels under reducing conditions (80 mM dithiothreitol) and transferred
onto polyvinylidene difluoride (PVDF) membranes using the MiniProteans System
(Bio-Rad Lab., Hercules, CA, USA). The blots were blocked and then probed with
anti-AGE MAb 6D12 (1:2500) for 60 min at room temperature. Protein bands were
visualized using enhanced chemiluminescence reagents and the densities of blots were
measured with a densitometer (Dolphin-Chemi mini System, Wealtec Corp., Sparks
Nevada, USA).
2-8. Measurement of lipid peroxidation
The samples of aorta or left ventricle (LV) were homogenized in RIPA buffer
(Sigma Chemical Co.) containing 1% protease inhibitor cocktail (Sigma Chemical Co.).
After centrifugation at 1600×g at 4℃ for 10 min, the supernatants were used for lipid
peroxidation assay. Lipid peroxidation was measured by the thiobarbituric acid reactive
substances (TBARS) of commercially available kits (Cayman Chemical Company, Ann
Arbor, Michigan, USA) and expressed as mM or nmole of malondialdehyde (MDA) per
mg gram of proteins. The fluorescence was measured at 540 nm with a
spectrophotometry.
2-9. Statistical analysis
Results are reported as the mean ± standard errors. Statistical two-way analysis of
variance was performed to compare the differences and to evaluate the effects on CRF
and OLM or PM on the physical properties of the rat arterial system. Simple-effect
analysis was used when significant interaction between CRF and OLM or PM occurred.
Differences among means within levels of a factor were determined using Tukey
honestly significant difference (HSD) method. P values less than 0.05 were considered
to be statistically significant.