Mechanisms underlying renoprotection during renin-angiotensin
system blockade
Maarten W.
Taal,
Glenn M.
Chertow,
Helmut G.
Rennke,
Anuradha
Gurnani,
Tang
Jiang,
Aliakbar
Shahsafaei,
Julia L.
Troy,
Barry M.
Brenner, and
Harald S.
Mackenzie
Renal Division, Department of Medicine, Brigham and Women's
Hospital, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
Potential determinants of chronic renal disease (CRD)
progression were studied in male Munich-Wistar rats subjected to 5/6 nephrectomy and treated with candesartan (Csn; n = 30)
or enalapril (Ena; n = 27) from 5 wk
postsurgery. Despite control of systolic blood pressure (SBP;
24 wk: Csn = 143 ± 9; Ena = 148 ± 8 mmHg), urinary protein excretion rates (UprV) increased over 24 wk
(Csn = 92 ± 10; Ena = 99 ± 8mg/day).
Glomerulosclerosis scores (GS) at 24 wk were similar for Csn (42 ± 7%) vs. Ena (42 ± 4%), values close to those of untreated
controls at 12 wk (43 ± 4%). At 24 wk, SBP and UprV
correlated strongly with GS, together accounting for 72% of the
variance in GS. Renal cortex mRNA levels (determined by competitive
RT-PCR) for transforming growth factor (TGF)-
1 and monocyte
chemoattractant protein (MCP)-1 were elevated in Csn and Ena at 12 wk
and remained higher at 24 wk vs. sham. Strong correlations were evident
among TGF-
1, MCP-1, and interleukin-1
and renal injury at 24 wk.
Cns and Ena are thus equally effective renoprotective agents in this
model. During renin-angiotensin system inhibition, renoprotection is
dependent on control of both SBP and UprV. Incomplete
suppression of renal cytokine gene expression may also contribute to
CRD progression.
angiotensin-converting enzyme inhibitor; angiotensin receptor
antagonist; glomerulosclerosis; systolic blood pressure; proteinuria; interleukin-1
; monocyte chemoattractant protein-1; transforming
growth factor-
 |
INTRODUCTION |
EARLY TREATMENT OF RATS
SUBJECTED to extensive renal mass ablation with
angiotensin-converting enzyme inhibitors (ACEI) effectively prevents
the focal and segmental glomerulosclerosis (FSGS) and tubulointerstitial fibrosis (TIF) that ensues in untreated rats, an
effect attributable, in part, to normalization of the raised glomerular
capillary hydraulic pressure (Pgc) characteristic of this
model of chronic renal disease (CRD) progression (3, 4). Because these experimental findings were later matched in landmark clinical trials (14, 27, 30, 40, 45, 46), ACEI therapy is
now widely regarded as a fundamental component of strategies designed
to retard the progression of CRD. Angiotensin subtype 1 receptor
antagonists (AT1RA), a novel class of antihypertensive drugs, inhibit the renin-angiotensin system (RAS) distal to
angiotensin-converting enzyme (ACE) and may offer therapeutic
advantages over ACEI (51). Nevertheless, previous studies
from this and other laboratories detected no significant differences in
the renoprotective effects of ACEI vs. AT1RA in
experimental models of CRD, when treatment was initiated before the
onset of substantial renal injury (2, 5, 19, 21, 26, 36, 41, 47,
58, 60). A single study, however, purports to show an advantage
of AT1RA over ACEI in 5/6 nephrectomized rats
(34). Notably, in this study treatment was initiated only
after renal injury was evident and was of considerably longer duration
than previous studies. These findings raised the possibility that
subtle benefits of AT1RA over ACEI may become evident only
over an extended time period in a model in which RAS blockade results
in a slowing but not an arresting of CRD progression. This is
particularly important because clinical trials of ACEI treatment in CRD
have also observed a slowing rather than an arresting of CRD
progression (14, 27, 30).
Because the therapeutic ideal is to arrest or even reverse CRD
progression, it is important to identify factors that may contribute to
the slow progression of CRD during RAS inhibition. Systemic blood
pressure has been shown in experimental (6) and clinical (20, 22, 23, 32, 37, 39) studies to be an important determinant of chronic renal injury, but the role of blood pressure in
the context of ACEI treatment requires further elucidation. Proteinuria, long regarded as a marker of glomerular injury, has recently been proposed as an important factor contributing to the
pathogenesis of CRD progression (42). Finally, recent
studies have found that extensive renal mass ablation provokes the
coordinated induction of several proinflammatory genes and infiltration
of the remnant kidney by macrophages (48, 53, 57).
These chronic inflammatory responses are prevented by early treatment
with ACEI or AT1RA, suggesting that macrophages and a
variety of proinflammatory molecules may contribute to the pathogenesis
of progressive renal fibrosis (53, 57). We hypothesized
that persistent upregulation of inflammatory and profibrotic gene
expression may also be a significant factor associated with slow
progression of renal injury during ACEI or AT1RA therapy.
In this study we utilized relatively large numbers of rats in a delayed
treatment protocol with prolonged follow up to 1) determine
whether, at doses that produce equivalent antihypertensive effects, the
AT1RA, candesartan, and the ACEI, enalapril, share equivalent renoprotective effects; and 2) examine the role
of systemic blood pressure and proteinuria as well as renal
inflammatory and profibrotic gene expression in contributing to the
slow progression of CRD during RAS inhibition.
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METHODS |
Animals.
Adult male Munich-Wistar rats (218-278 g) were obtained from
Simonsen Laboratories (Gilroy, CA), housed under standard conditions, and given unrestricted access to standard rodent chow and water. Rats
were subjected to either renal mass ablation by right nephrectomy and
ligation of two or three branches of the left renal artery, producing
infarction of approximately two-thirds of the left kidney (n = 63), or sham operation by laparotomy and
mobilization of the renal vessels (Sham rats; n = 15).
All surgical procedures were performed under pentobarbital anesthesia
(Nembutal, 50 mg/kg ip; Abbott Laboratories, Chicago, IL). At 2-wk
intervals, systolic blood pressure (SBP) was measured by the tail-cuff
method, and daily urinary protein excretion rate
(UprV) was determined on urine collected from rats
individually housed in metabolic cages for 24 h. At 5 wk after
renal mass ablation, rats were stratified according to SBP and
UprV and allocated to the following groups. Csn rats
(n = 30) received candesartan cilexetil [TCV-116;
Takeda Chemical Industries, Osaka, Japan; 50 mg/l (3-7
mg · kg
1 · day
1) in the
drinking water]. Vehicle comprising ethanol (0.1%, vol/vol), polyethylene glycol (0.1%, vol/vol), and sodium bicarbonate (5 mmol/l)
was added to achieve water solubility of candesartan. Ena rats
(n = 27) received enalapril [Merck Research
Laboratories, Rahway, NJ; 110 mg/l (7-15
mg · kg
1 · day
1) in the
drinking water with sodium bicarbonate (5 mmol/l)]. Some dosage
adjustments were made initially to achieve equivalent blood pressure
control in the treatment groups. Six rats were killed at 5 wk after
renal ablation, and the remnant kidneys were harvested to provide
pretreatment data (5WK rats; n = 6). The remaining rats
were studied for a total of 12 (set A: CsnA,
n = 11; EnaA, n = 11;
ShamA, n = 6) or 24 wk (set B:
CsnB, n = 19; EnaB,
n = 16; ShamB, n = 9). At
the end of the observation period, rats were anesthetized with
pentobarbital, and portions of renal cortex distant from the infarct
scar were excised and snap frozen in liquid nitrogen for subsequent RNA
extraction and immunohistology. The remnant kidney was then
perfusion-fixed with 1.25% glutaraldehyde in 0.1 mmol/l sodium
cacodylate buffer (pH 7.4), delivered through a catheter in the
abdominal aorta at the measured SBP of each rat. Kidneys were weighed
after perfusion fixation. To evaluate renal hypertrophy, final remnant
kidney weight, corrected for the increase in weight associated with
perfusion fixation, was compared with an estimate of baseline remnant
kidney weight taken as one-third of the weight of the right kidney
removed at the time of renal mass ablation. The correction factor
(1.38) was derived by comparing the weights of the unfixed right kidney
with those of the perfusion-fixed left kidneys removed
contemporaneously from 15 sham-operated rats.
Untreated controls were not included in the study because, on the basis
of previous experience in our laboratory, the chance for survival to 24 wk was expected to be close to zero. For purposes of comparison, data
from untreated control rats after 5/6 nephrectomy were pooled from
previous 12-wk studies recently performed in this laboratory. The
percentage of glomeruli affected by sclerosis in 22 untreated rats at
12 wk after 5/6 nephrectomy was 43 ± 17 (SD) % (29,
36).
Morphology.
Renal tissue was postfixed in 10% phosphate-buffered Formalin,
embedded in paraffin, and processed for light microscopy. The frequency
of FSGS was estimated by examining all glomeruli seen in one or two
coronal sections from each kidney stained by the periodic acid-Schiff
method. Segmental sclerosis was defined as glomerular capillary
collapse with hyaline deposition and/or adhesion to the parietal layer
of Bowman's capsule. A glomerulosclerosis score (GS) was determined by
expressing the number of glomeruli with segmental or global sclerosis
as a percentage of the total number of glomeruli counted for each rat
(mean 132; range 73-274/rat). Tubulointerstitial injury, as
evidenced by dilated tubules containing protein casts and interstitial
inflammation or fibrosis, was assessed at medium power on the same
sections before evaluation of the glomerulosclerosis. A scoring system
[tubulointerstitial score (TIS)] was used to grade the injury from 0 to 3 on the basis of the percentage of abnormal tissue (0, <20,
20-50, and >50%, respectively). The pathologist was unaware of
the group assignment of individual rats.
Chemical analysis.
The concentration of protein in the urine was determined by
spectophotometry after precipitation with 3% sulfosalicylic acid.
Competitive RT-PCR.
Total RNA was extracted from frozen portions of renal cortex by the
cesium chloride ultracentrifugation method (11). RNA was
quantitated by determination of ultraviolet absorbance at 260 nm, and
its purity was assessed by measuring the optical density ratio at 260 and 280 nm. For preparation of cDNAs, 4 µg of heat-denatured RNA were
used in an RT reaction. The entire sample in a total volume of 20 µl
contained 4 µg of RNA; 0.5 mM each of dATP, dCTP, dGTP, and dTTP
(Pharmacia Biotech, Piscataway, NJ); 0.5 µg oligo-d(T)12-18 (Pharmacia Biotech); 40 U RNasin ribonuclease inhibitor (Promega, Madison, WI); and 200 U Moloney murine leukemia virus RT (Life Technologies, Gaithersburg, MD) in a buffer of (in mM) 50 Tris · HCl (pH 8.3), 75 KCl, 3 MgCl2, and 10 dithiothreitol. The solution was incubated for 60 min at 37°C and
then held at 95°C for 5 min to arrest the reaction.
Preparations of cDNA were then used as substrate for competitive PCR
reaction by using competitive DNA mimics and oligonucleotide primer
sets (Genosys Biotechnologies, Woodlands, TX). Competitive DNA mimics
for each factor, comprising a segment of neutral DNA with sequences
complimentary to the gene-specific primers attached to each end, were
constructed by using a PCR MIMIC construction kit (Clontech
Laboratories, Palo Alto, CA). Primer sets were designed for rat
transforming growth factor (TGF)-
1, monocyte chemoattractant protein
(MCP)-1, interleukin (IL)-1
, and
-actin on the basis of published
cDNA sequences (21). An equal volume of each cDNA solution
was used for amplification in 20 µl of reaction mixture containing
competitive DNA mimic, 0.5 µM primer sets; 0.5 U Taq DNA
polymerase (Pharmacia Biotech); 250 µM each of dATP, dCTP, dGTP, and
dTTP (Pharmacia Biotech) in a buffer of 10 mM Tris · HCl (pH
9.0) and optimal concentration of MgCl2. PCR was performed by using a Peltier Thermal Cycler (MJ Research, Watertown, MA). Optimal
PCR conditions, namely, concentration of competitive DNA mimic,
annealing temperature, and amplification cycles, were determined for
each factor in preliminary studies. Amplification was initiated with
incubation at 94°C for 2 min followed by amplification cycles as
follows: 94°C for 15 s, annealing temperature for 30 s, and 72°C for 1 min. Sequences of oligonucleotide primer sets and optimal conditions are listed in Table 1. PCR
products (7 µl) were subjected to gel electrophoresis (5%
polyacrylamide), and then DNA bands were visualized under ultraviolet
light after ethidium bromide staining (0.05 µg/ml for 10 min) and
photographed (10). Densities of competitive mimic and
target gene DNA bands were measured by scanning densitometry by using
ScanJet 4c (Hewlett Packard, Corvallis, OR) with National Institutes of
Health Image software. The ratios of the densities of the respective
bands were plotted to establish a linear relationship of the ratios
over serial dilutions of template (Fig.
1, A and B). Thus
absolute amounts of RNA from unknown samples were calculated as
previously described (21, 55) from the known amount of the
mimic in the starting reaction by using the formula
where
is the gradient of the log plot of target gene
product-mimic product vs. serial dilutions of starting cDNA (Fig. 1B). Specimens were run in duplicate, and the average value
was used. We have previously established that this assay is readily capable of detecting a twofold difference in target gene concentration (21, 53). As the number of specimens exceeded the capacity of the thermal cycler, all specimens from the study could not be
included in a single PCR reaction. Specimens from 5WK,
CsnA, EnaA and Shama rats were
therefore included in one set of PCR reactions and specimens from 5WK,
CsnB and Enab, in a separate set of PCR
reactions. To allow direct comparison of results from different PCR
reactions, data were expressed as ratios to the mean value for the 5WK
group.
-Actin mRNA levels were used to confirm that starting amounts
of cDNA were similar among groups.

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Fig. 1.
A: polyacrylamide gel showing electrophoretic
bands for mimic and gene-specific [transforming growth factor
(TGF)- 1] PCR products at different amounts of starting cDNA.
B: log-log plot of starting cDNA amount vs. the ratio of
gene-specific (TGF- 1) to mimic PCR product concentration (in
duplicate) showing a linear relationship over serial dilutions of
starting cDNA. C: representative portion of a gel showing
electrophoretic bands for mimic and gene-specific (TGF- 1) PCR
products for different samples of renal cortex mRNA. Specimens in
a and b were from rats who had low levels of
glomerular injury after 24 wk [glomerulosclerosis scores (GS) = 24.2 and 18.0%, respectively], whereas specimen in c was
from a rat with severe glomerular injury at 24 wk (GS = 75.6%).
Higher levels of TGF- 1 mRNA expression can be appreciated in the
latter even on visual inspection.
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Immunohistochemistry.
Expression of TGF-
1, MCP-1, and IL-1
proteins and macrophage
infiltration was assessed by immunohistochemistry. For macrophage staining, 4-µm paraffin sections of fixed tissues were used for immunoperoxidase analysis after baking at 60°C for 1 h,
deparaffinization, and rehydration (xylene × 4 for 3 min each,
100% ethanol × 4 for 3 min each, and running water for 5 min).
The sections were then microwave treated at 93°C for 30 min in
preheated 10 mM citrate buffer, pH 6.0, cooled for 15 min, and
transferred to PBS. Sections were then blocked (for 15 min) with a
1.5% solution of serum from the animal source of the secondary
antibody at room temperature. Next, sections were incubated with mouse
monoclonal antibodies to the monocyte/macrophage marker ED1 (clone ED1,
1:150 dilution, Biosurface International, Camarilla, CA) for 1 h
in a humid chamber at room temperature. The secondary antibody
(Vectastain Elite ABC Kit, Vector Laboratories, Burlington, CA) was
used according to manufacturer's instructions. Slides were rinsed with
PBS between each incubation. Sections were developed by using
3,3'-diaminobenzidine (Sigma, St. Louis, MO) as substrate and
counterstained with Gill's hematoxylin (Fisher Scientific, Pittsburgh,
PA). Macrophage infiltration was assessed by counting the number of
ED1-positive cells in 10 glomerular profiles and in 10 randomly chosen
0.25 × 0.25-mm areas of tubulointerstitium for each kidney.
For TGF-
1, MCP-1, and IL-1
staining, 4-µm sections of frozen
tissues were fixed in acetone for 10 min at
20°C and then rinsed
with PBS. Sections were then blocked with a 1.5% solution of serum and
incubated with primary antibodies, hamster anti-mouse antibody against
MCP-1 (clone 2H5, 1:50 dilution, Biosurface International), rabbit
polyclonal antibodies against IL-1
(1:100 dilution, Endogen, Woburn,
MA), and TGF-
1(V) (1:150 dilution, Santa Cruz Biotechnology, Santa
Cruz, CA), and then secondary antibodies as described above. Methyl
green was used as a counterstain.
Statistical analysis.
Continuous variables, expressed as means ± SE, were compared with
ANOVA derived from general linear models. Pairwise comparisons of
physiological data from weeks 4, 12, and 24 were
performed by using the Student-Neuman-Keuls procedure. Determinants of
proteinuria, glomerulosclerosis, and tubulointerstitial injury were
analyzed by using multivariable linear regression with stepwise
variable selection. Multiplicative interaction terms were tested to
evaluate whether the estimated effects of blood pressure and degree of proteinuria on glomerulosclerosis were uniform across treatment modality. Dependent variable distributions approximated the normal, and
regression diagnostics showed no outliers. Repeated-measures ANOVA,
factorial ANOVA, and paired t-tests were employed for other comparisons. For PCR data, which were not normally distributed, differences among multiple groups were assessed by using the
Kruskal-Wallis test and those between two groups with the Mann-Whitney
U-test. P < 0.05 were considered
significant. Statistical analyses were conducted by using Statview 4.01 (Abacus Concepts, Berkley, CA) and SAS 6.08 (SAS Institute, Cary, NC).
 |
RESULTS |
Chronic studies.
Mean body weight increased in all groups during the study, and no
statistical differences in body weight developed between candesartan-
and enalapril-treated rats over time in either the 12- or 24-wk sets.
Sham-operated rats attained significantly greater body weight than
partially nephrectomized rats in the pretreatment period and continued
to maintain significantly higher average body weight than
enalapril-treated rats in the 24-wk set. In the 12-wk set only the
difference between sham-operated and enalapril-treated rats in the
pretreatment period was statistically significant. (Table
2).
Mean SBP increased in all groups after partial nephrectomy and did not
differ statistically among the groups before initiation of therapy at
week 5. Treatment with either candesartan or enalapril resulted in an initial fall in SBP to levels similar to those of
sham-operated rats. SBP remained similar among treated groups and
sham-operated rats over weeks 6- 12 in both 12- and 24-wk sets. Thereafter, there was a gradual increase in SBP such
that from 18 wk, SBP levels were statistically higher than the lowest values, observed at 8 wk, in both CSNB and ENAB
rats. There were no statistically significant differences in SBP
between treatment groups over time, in either 12- or 24-wk sets (mean
differences: CsnA vs. EnaA = 11 mmHg,
P = 0.26; CsnB vs. EnaB = 7 mmHg, P = 0.41). Sham-operated rats remained
normotensive throughout the study (Figs.
2A and
3A). UprV
increased after partial nephrectomy and was similar among the groups
before initiation of therapy at week 5. In both candesartan-
and enalapril-treated rats, UprV declined at first, but
later increased progressively to reach levels approximately twofold
those of pretreatment values and eight- to ninefold those of
sham-operated rats at 24 wk. No statistically significant differences
were observed in UprV between treatment groups over time,
in either the 12- or 24-wk sets (mean differences: CsnA vs.
EnaA = 8.8 mg/day, P = 0.16;
CsnB vs. EnaB = 8.4 mg/day, P = 0.31). In sham-operated rats, mean UprV
remained low, although a small increase was evident with time (Figs.
2B and 3B). UprV was directly
correlated with SBP in combined data from CsnB and EnaB rats at 12 and 24 wk (r = 0.60 and
0.73, respectively; P < 0.0001 for both). There were
still no differences in UprV between the treatment groups
after adjusting for the effects of SBP (P = 0.50 and
P = 0.70 at 12 and 24 wk, respectively). Furthermore, there was no effect of treatment group on the relationship between UprV and SBP (interactive terms: P = 0.78 and P = 0.30 at 12 and 24 wk, respectively).

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Fig. 2.
A: systolic blood pressures (SBP; means ± SE) of rats in the 12-wk set over time. SBP increased in both groups
after partial nephrectomy and did not differ statistically between them
before initiation of therapy at week 5. Treatment with
either candesartan (CsnA; ) or enalapril
(EnaA; ) resulted a fall in SBP to levels
similar to those of sham-operated rats (ShamA;
). There were no statistically significant differences
in SBP between CsnA and EnaA over weeks
6-12 (on treatment). Sham-operated rats remained normotensive
over 12 wk. B: urinary protein excretion rate
(UprV) of rats in the 12-wk set over time. UprV
increased in both groups after partial nephrectomy and did not differ
statistically between them before initiation of therapy at week
5. Treatment with either candesartan (CsnA;
) or enalapril (EnaA; ) was
associated with an initial decline in UprV, which later
increased from week 8 to week 12. There were no
statistically significant differences in UprV between
CsnA and EnaA rats over weeks
6-12. UprV remained at normal levels in
sham-operated rats over 12 wk (ShamA; ).
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Fig. 3.
A: SBP (means ± SE) of rats in the 24-wk
set over time. SBP increased in both groups after partial nephrectomy
and did not differ statistically between them before initiation of
therapy at wk 5. Treatment with either candesartan (CsnB;
) or enalapril (Enab; )
resulted in an initial fall in SBP to levels similar to those of
sham-operated rats (ShamB; ). However, SBP
increased gradually over time such that from 18 wk, levels were
significantly higher than the lowest values, observed at 8 wk in both
CsnB and EnaB. There were no statistically
significant differences in SBP between CsnB and
EnaB over weeks 6-24 (on treatment).
Sham-operated rats remained normotensive over 24 wk. B:
UprV of rats in the 24-wk set over time. UprV
increased in both groups after partial nephrectomy and did not differ
statistically between CsnB and EnaB before
initiation of therapy at week 5. Treatment with either
candesartan (CsnB; ) or enalapril
(EnaB; ) was associated with an initial
decline in UprV followed by a progressive increase over
weeks 8-24. There were no statistically significant
differences in UprV between CsnB and
EnaB rats over weeks 6-24. UprV
remained at normal levels in sham-operated rats over 24 wk
(ShamA; ).
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Remnant kidneys hypertrophied considerably in all groups such that the
weight increased three to fourfold over baseline. There were no
statistically significant differences in final remnant kidney weight
among the groups. Analysis of remnant kidney weights, expressed as
kidney weight-to-body weight ratio, yielded similar results (Table
3).
Morphology.
Histological data are summarized in Table
4. At 5 wk after partial nephrectomy and
before initiation of therapy, glomerulosclerosis was evident in a mean
of 26 ± 6% of glomeruli (5WK rats). At 12 wk postsurgery, the
extent of glomerulosclerosis in candesartan- and enalapril-treated rats
was similar to that observed before treatment in the 5-wk group;
moreover, there was no statistical difference in mean GS between
CsnA and EnaA. At 24 wk after surgery, there
was again no statistically significant difference in the mean GS of
CsnB vs. EnaB rats. Comparison of combined
CsnB and EnaB data with combined
CsnA and EnaA data revealed a trend toward more
extensive glomerulosclerosis at 24 vs. 12 wk (P = 0.06 by ANOVA). When these data are viewed in the context of data for untreated 5/6 nephrectomized controls at 12 wk, it is evident that both
treatments slowed the progression of secondary FSGS such that the
extent of glomerulosclerosis previously observed at 12 wk after 5/6
nephrectomy [GS = 43 ± 17 (SD) %], was attained only
after 24 wk in CsnB and EnaB rats (Fig.
4). Minimal glomerulosclerosis was noted
in sham-operated rats.

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Fig. 4.
GS at different time points in untreated (5WK; solid
bar), Csn (hatched bars), and Ena rats (stippled bars). Horizontal
lines indicate means ± SD for GS at 12 wk after 5/6 nephrectomy
in 22 untreated control rats from previous studies.
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Tubulointerstitial injury showed similar patterns of change to those of
glomerulosclerosis and was also not statistically different between CSN
and ENA rats at 12 or 24 wk. There was a direct and highly significant
correlation between GS and TIS in pooled data from CsnB and
EnaB rats (r = 0.85; P < 0.001). As with glomerulosclerosis, minimal tubulointerstitial injury
developed in sham-operated rats.
Multivariable analysis.
Analysis of data from rats killed at 5 wk after surgery revealed
statistically significant correlations between pretreatment UprV and GS (r = 0.87; P = 0.02) or TIS (r = 0.87; P = 0.02). There were no statistically significant correlations between
pretreatment SBP and GS or TIS.
At 24 wk, direct and highly significant correlations were evident
between SBP and GS (r = 0.81; P < 0.0001) (Fig. 5A). Similarly, UprV was highly correlated with GS (r = 0.86; P < 0.0001) (Fig. 5B). By contrast,
there was no effect of treatment group on GS at 24 wk
(P = 0.9). Stepwise multiple linear regression analysis with GS as the dependent variable and SBP, UprV, and
remnant kidney weight-to-body weight ratio as independent variables,
entered only SBP and UprV into the model. These variables
together accounted for 72% of the variance in glomerulosclerosis
observed at 24 wk. The magnitude of the effects of SBP and
UprV as determinants of glomerulosclerosis was such that a
10-mmHg change in SBP or a 10 mg/day change in UprV was
each associated with a change of three percentage points in GS at 24 wk.

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Fig. 5.
A: scatterplot for GS vs. SBP at 24 wk in
CsnB ( ; dashed line, regression line) and
EnaB ( ; dotted line, regression line) rats.
Linear regression equation for combined data (solid line):
y = 34.5 + 0.52x;
r2 = 0.65. B: scatterplot for GS
vs. UprV at 24 wk in in CsnB ( ;
dashed line, regression line) and EnaB ( ;
dotted line, regression line) rats. Linear regression equation for
combined data (solid line): y = 6.5 + 0.51x; r2 = 0.74.
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TIS at 24 wk also correlated with SBP and UprV
(r = 0.78 and 0.69, respectively; P < 0.0001 for both). Stepwise multiple linear regression analysis with TIS
as the dependent variable and the same independent variables as above
entered only SBP into the model.
Competitive RT-PCR.
Mean levels of
-actin mRNA were similar among groups for each set of
PCR reactions, confirming that starting concentrations of cDNA were
similar and not subject to systematic error. Renal cortex mRNA levels
for TGF-
1 and MCP-1 in 5/6 nephrectomized rats before treatment were
about twofold higher than those of sham-operated rats. At 12 wk after
surgery, TGF-
1 and MCP-1 mRNA levels were similar to pretreatment
5WK values in both CsnA, and EnaA rats and were
significantly higher than in ShamA rats. For IL-1
, mRNA
levels were similar in 5WK and ShamA rats. At 12 wk, IL-1
mRNA exhibited a trend toward higher levels in
CsnA, and EnaA vs. ShamA rats that
was not statistically significant. At 24 wk after 5/6 nephrectomy,
TGF-
1 mRNA levels were significantly lower than pretreatment values
in CsnB and EnaB rats but were still
significantly higher than those of sham-operated rats. By contrast,
MCP-1 mRNA levels were not different from pretreatment values in
CsnB and EnaB rats and remained higher than
ShamA levels. IL-1
mRNA levels in both CsnB
and EnaB rats were similar to those of ShamA
rats (Fig. 6). There were no
statistically significant differences in mRNA levels for any of the
genes examined between CSN and ENA rats at either 12 or 24 wk.

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Fig. 6.
Renal cortex mRNA levels for TGF- 1 (top),
monocyte chemoattractant protein (MCP)-1 (middle), and
interleukin (IL)-1 (bottom; expressed as a ratio to the
mean 5-wk untreated value) in 5/6 nephrectomized rats receiving no
treatment (5WK; solid bars), Csn (hatched bars), Ena (stippled bars),
and sham-operated rats (Sham; open bars). *P < 0.05 vs. 5WK. P < 0.05 vs. Sham. §P = 0.05 vs. Sham.
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Because there were no differences between candesartan- and
enalapril-treated rats with respect to any of the parameters determined in this study, data from both groups were pooled for further analysis. In this combined group, strong and highly statistically significant correlations were evident between 24-wk renal cortex mRNA levels for
TGF-
1, MCP-1, and IL-1
and the extent of FSGS (Fig.
7) or TIF (Table
5). Somewhat weaker but nevertheless
statistically significant correlations were evident among TGF-
1,
MCP-1, and IL-1
, and SBP or UprV (Table 5). Analysis of
pooled CsnA, and EnaA data revealed only weak
or absent correlations among these parameters at 12 wk after surgery
(data not shown).

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Fig. 7.
Scatterplots of renal cortex mRNA levels for TGF- 1
(top) MCP-1 (middle), and IL-1
(bottom) vs. GS for pooled data from Csn and Ena rats
(n = 34) at 24 wk. Regression lines and correlation
coefficients are also shown.
|
|
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|
Table 5.
Correlation coefficients for renal cortex cytokine mRNA levels vs.
measures of renal injury in a combined group of Csn and Ena
rats at 24 wk after 5/6 nephrectomy
|
|
Immunohistology.
Immunohistology confirmed that the increases in gene expression
detected by competitive RT-PCR were accompanied by qualitative increases in expression of the gene product and localized the protein
expression within the renal cortex. Negative controls, in which no
primary antibody was used, showed minimal staining of tubules and no
glomerular staining. For TGF-
1, kidneys from sham-operated rats
exhibited moderate staining of tubules and negative staining of
glomeruli. Among 5WK rats, positive TGF-
1 staining of both tubules
and glomeruli was observed. Patterns of staining similar to those of
5WK rats were observed among rats in both treatment groups, with
particularly strong staining seen in areas of segmental
glomerulosclerosis (Fig. 8,
A-C). MCP-1 staining was limited to minimal
positivity of tubule cells in sham-operated rats. Positive MCP-1
staining of tubules and glomeruli was observed in 5WK rats and among
rats from both treatment groups (Fig. 8,
D-F). Positive staining for IL-1
was
localized mainly to tubule cells in specimens from all groups. However,
among 5WK rats and rats from the treatment groups, focal areas of
positive staining for IL-1
were observed in some glomeruli (Fig. 8,
G-I). (This observation is consistent with
staining of individual cells within glomeruli but, due to the
limitations of histology performed on frozen tissue sections, this
could not be confirmed.)

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Fig. 8.
Immunohistology. A-C: TGF- 1
immunostaining was positive only in tubule cells of Sham rats
(A; ×100 magnification) whereas tubule and glomerular
staining was evident in 5WK rats (B; ×100 magnification)
and in rats from both treatment groups (C; animal from
CsnB shown, ×100 magnification). Particularly strong
staining was observed in glomeruli exhibiting extensive sclerosis.
D-F: MCP-1 immunostaining was minimal in
tubules and negative in glomeruli from Sham rats (D; ×40
magnification). Among 5WK rats (E; ×100 magnification) and
rats from both treatment groups (F; animal from
CsnB shown, ×100 magnification), positive staining of
tubules and glomeruli was observed. G-I:
IL-1 immunostaining was localized mainly to tubule cells among Sham
(G; ×100 magnification), 5WK (H; ×100
magnification), and treated rats (I; animal from
CsnB shown, ×100 magnification). However, among 5WK and
treated rats, focal areas of positive staining, consistent with
staining of individual cells, were observed in some glomeruli
(H and I).
|
|
Macrophage infiltration.
Extensive infiltration of glomeruli and remnant kidney interstitium by
macrophages was evident before initiation of therapy at 5 wk after
surgery. Whereas macrophages were virtually absent from the kidneys of
sham-operated rats, a mean of 5.7 ± 0.22 macrophage/glomerular profile and 6.6 ± 0.19 macrophages/0.0625-mm2 area of
interstitium were observed in 5WK rats. Treatment with candesartan or
enalapril was associated with two- to fivefold reductions in glomerular
macrophage infiltration and an approximately fourfold reduction in
interstitial macrophages at 12 wk after surgery. Nevertheless, the
extent of macrophage infiltration of both glomeruli and interstitium
remained significantly higher in treated rats vs. sham-operated rats at
12 and 24 wk. Glomerular macrophage counts were slightly, albeit
significantly, higher in enalapril- vs. candesartan- treated rats at 12 and 24 wk. There was no difference in interstitial macrophage counts
among treatment groups at either time point (Fig.
9).

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Fig. 9.
Glomerular (top) and interstitial
(bottom) ED1-positive cell (macrophage) counts in 5/6
nephrectomized rats receiving no treatment (5WK; solid bars), and Csn
(hatched bars), Ena (stippled bars), and Sham rats (open bars).
*P < 0.05 vs. 5WK. P < 0.05 vs.
Sham. §P < 0.05 vs. Csn.
|
|
 |
DISCUSSION |
These results show that, even when started after the onset of
renal injury, the renoprotective effects of candesartan are equivalent
to those of enalapril when dosing is adjusted to achieve similar levels
of SBP control. Comparison with data for untreated rats from previous
studies in this model shows that delayed treatment with either ACEI or
AT1RA, appears to slow the rate of progression of
glomerulosclerosis by about one-half, an effect reminiscent of that
achieved in clinical trials (14, 27, 30). Our study differs from the previous study comparing delayed therapy with ACEI and
AT1RA in the 5/6 nephrectomy model by virtue of its longer duration and increased statistical power (34).
Furthermore, 5/6 nephrectomy was achieved by surgical excision of renal
mass in the former study whereas we performed infarction of 2/3 of the
left kidney, a model resulting in more severe renal injury and greater
activation of the RAS (12, 54) and therefore more likely
to expose subtle differences in efficacy between ACEI and AT1RA. Nevertheless, we detected no differences among the
treatment groups with respect to any of the markers of renal injury
examined in this study (UprV, GS, and TIS) or in the
remnant kidney levels of cytokine gene expression. Moreover, no
tendencies for differences to emerge were detected even over a
prolonged observation period. The extent of hypertrophy of the remnant
kidney also was similar between ACEI- and AT1RA-treated
rats. Finally, the correlations among SBP, UprV, and GS as
assessed by linear regression techniques were not affected by treatment
group. These data therefore provide the most conclusive evidence to
date that ACEI and AT1RA are equivalent in their
renoprotective effects in this model of progressive CRD.
On the basis of the differences in the mechanisms, whereby ACEI and
AT1RA inhibit the RAS, it has been suggested that
AT1RA may have therapeutic benefits over ACEI because they
inhibit the actions of ANG II formed by other serine proteases in the
presence of ACEI or because of the antihypertensive and
antiproliferative effects that may result from stimulation of
AT2 receptors by elevated ANG II levels during blockade of
AT1 receptors. On the other hand, it has been suggested
that at least some of the therapeutic benefit of ACEI results from
elevation of bradykinin levels, which is not present during
AT1RA treatment (51). Our findings strongly suggest that although they inhibit the RAS at different levels, both
ACEI and AT1RA exert their beneficial effects predominantly by inhibiting the effects of ANG II mediated by AT1
receptors. This conclusion is consistent with the findings of previous
studies showing that the elevated bradykinin levels associated with
ACEI (2, 24, 33), or the AT2 receptor
stimulation that results indirectly from blockade of the
AT1 receptor (9), do not appear to play
significant renoprotective roles.
The success of RAS inhibitors in reducing renal injury in
nonhypertensive models of renal disease (17) and
preserving renal function in normotensive patients (27)
suggests that functional integrity of the RAS may itself be regarded as
a risk factor for CRD progression. On the other hand, RAS inhibitors
are highly effective antihypertensive and antiproteinuric agents. It is
not yet clear to what extent blood pressure control retains its
importance as a renoprotective measure in the context of RAS
inhibition. Nor is it clear whether reduction of proteinuria merely
reflects blood pressure reduction or if lower levels of protein
excretion at a given level of systemic blood pressure are associated
with additive renal protective effects. A major finding of this study, therefore, is the demonstration by linear regression techniques that
systolic blood pressure and urinary protein excretion are independent
determinants of glomerulosclerosis in rats receiving inhibitors of the
RAS after extensive renal ablation. Together, SBP and UprV
accounted for 72% of the variance in GS, implying that they represent
the major determinants of glomerulosclerosis in this model.
Although it is not possible to exclude from these data the possibility
that higher levels of blood pressure merely reflect greater severity of
renal injury, it is true to say that failure to control blood pressure
optimally in this model was associated with failure to achieve renal
protection. Furthermore, the lack of any correlation between the level
of SBP and severity of renal injury at an early time point, before the
initiation of treatment, argues in favor of a direct effect of blood
pressure on renal disease progression over time. Using radiotelemetry,
Bidani et al. (6) also found a strong correlation
(r = 0.88) between mean SBP and glomerulosclerosis in
untreated 5/6 nephrectomized rats. In keeping with our observations,
the correlation between SBP and glomerular injury was much weaker
during the first 2 wk after injury. The renoprotective effect of
lowering blood pressure has been clearly established in clinical
studies of CRD (20, 22, 23, 32, 37, 39). In diabetic
patients treated with ACEI, lower target levels of blood pressure
control have been shown to result in greater renoprotective effects in
one randomized trial (28). Our data are therefore
consistent with this clinical evidence that the level of blood pressure
control remains an important determinant of progressive renal injury in
CRD during treatment with inhibitors of the RAS. It should be
remembered that micropuncture studies in 5/6 nephrectomized rats
suggest that Pgc, rather than systemic blood
pressure per se, is the critical determinant of renal injury (4,
50) and that ACEI (4) and AT1RA
(26, 29) reduce both systemic and glomerular capillary pressures.
Proteinuria has traditionally been regarded merely as a marker of
glomerular injury. Recent clinical studies, however, report that
proteinuria may also be an independent epidemiological risk factor of
CRD progression (8, 14, 39). Furthermore, treatments that
reduce proteinuria also slow the progression of CRD (14, 27,
30), and a reduction in proteinuria, independent of blood pressure, was associated with slower progression of CRD in the MDRD
study (39). Together, these findings raise the possibility that proteinuria per se exacerbates renal injury. Experimental observations suggest mechanisms whereby filtered proteins may contribute to renal damage. Exposure of mesangial cells to plasma lipoproteins in vitro results in proliferation, expression of proinflammatory cytokines, and synthesis and elaboration of
extracellular matrix protein, all of which may contribute to the
pathogenesis of glomerulosclerosis (15, 44). More
recently, culture of tubular epithelial cells in the presence of a
variety of plasma proteins has been shown to induce production of
proinflammatory cytokines and extracellular matrix proteins (1,
56, 59, 61), responses that may contribute to tubulointerstitial
fibrosis. In vivo, proteinuria induced by protein overload was
associated with renal expression of cell adhesion molecules and
chemoattractants, resulting in interstitial inflammation and fibrosis
(13). A meta-analysis of 57 animal studies, including
various models of renal disease, reported consistent positive
associations between the level of protein- or albuminuria and the
severity of glomerulosclerosis (mean weighted correlation coefficients
r = 0.82 and 0.76) (38). We have confirmed
that a direct correlation exists between proteinuria and glomerular
injury in 5/6 nephrectomized rats receiving RAS inhibitors
(r = 0.86), independent of the level of blood pressure. This implies that at a given level of blood pressure, rats with higher
levels of proteinuria can be expected to develop more severe renal
injury, a conclusion similar to those supported by clinical studies
(14, 39). Although these data do not prove that
proteinuria per se contributes to renal injury, they do reveal the
extent to which the renoprotective effects of ACEI and
AT1RA are related to their antiproteinuric effects.
The detection of inflammatory and profibrotic gene induction and
macrophage infiltration in the remnant kidney supports the notion that
inflammatory processes may contribute to the progressive renal injury
and fibrosis that follows 5/6 nephrectomy. Among possible mechanisms
whereby proinflammatory gene expression may be stimulated in the
remnant kidney are exposure of glomerular cells to mechanical stresses
resulting from augmented glomerular hemodynamics (35, 43,
49), direct effects of ANG II (18, 25), and
exposure of tubule epithelial cells to abnormal amounts of filtered
protein (13, 52, 56, 59). Thus components of an
inflammatory process may be induced in the remnant kidney in the
absence of classic immune stimuli. Previous studies from this and other
laboratories have shown that the protection from progressive renal
injury afforded by ACEI or AT1RA treatment initiated early
after 5/6 nephrectomy is associated with normalization of Pgc (4, 29), suppression of proinflammatory
gene induction to levels similar to those of sham-operated rats, and
inhibition of renal macrophage infiltration to levels only slightly
greater than sham (53, 57). In this study, we observed
that when treatment was delayed until 5 wk after 5/6 nephrectomy, a
time point when remnant kidney mRNA levels for TGF-
1 and MCP-1 are
known to be upregulated (53), ACEI or AT1RA
did not suppress the expression of these two cytokines to normal
levels. At 12 wk postsurgery mRNA levels for TGF-
1 and MCP-1 were
similar to those observed before the initiation of treatment and
remained significantly higher than those of sham-operated rats. At 24 wk after surgery, TGF-
1 mRNA levels were significantly lower than
pretreatment values but remained significantly higher than sham levels,
and MCP-1 mRNA levels remained at pretreatment values. Thus failure of
suppression of the TGF-
1 and MCP-1 responses at 12 wk, when renal
injury had not yet progressed beyond that observed before the
initiation of treatment, was associated with slow progression of renal
injury despite likely amelioration of adverse glomerular hemodynamic
factors. IL-1
mRNA levels were not elevated in pretreatment vs.
sham-operated rats. This is consistent with previous observations from
this laboratory that IL-1
induction was not apparent until 8 wk
after 5/6 nephrectomy (53). The trend toward higher
IL-1
mRNA levels in both treatment groups vs. sham at 12 wk suggests that failure of suppression of this gene, a product of activated macrophages, may also be associated with subsequent progression of
injury. The strong correlations observed between the extent of renal
injury at 24 wk (as measured by either GS or TIS) and mRNA levels for
TGF-
1, MCP-1, and IL-1
further support the hypothesis that
upregulation of these proinflammatory and profibrotic genes contributes
to progressive renal injury.
Together, these observations in remnant kidneys indicate 1)
that incomplete suppression of proinflammatory gene expression with
ACEI or AT1RA treatment is associated with failure to
arrest the progression of renal injury and 2) that the
extent of progression is directly correlated with the level of gene
expression. It should be stressed that these observations were made in
rats receiving chronic treatment at doses of ACEI or AT1RA
with documented success in normalizing Pgc even when
initiated after the onset of renal injury in this model (16, 26,
31). This implies that the process of renal injury initiated by
glomerular capillary hypertension, and the direct or indirect effects
of ANG II, eventually may be sustained more by autonomous cellular and
molecular factors, and become less dependent on the initiating factors.
This notion is consistent with the observations of Ichikawa and others
(16) that in glomeruli with severe established injury,
treatment with enalapril did not prevent further progression to global
sclerosis (16). Alternatively, it remains possible that,
in the face of existing renal injury, treatment with ACEI or
AT1RA did not completely normalize Pgc in the
long term or achieve total blockade of the RAS. In keeping with
suggestions by other authors (7), these findings imply
that patients in whom progression of chronic renal injury persists,
albeit slowly, during RAS blockade, may benefit from additional therapy
targeting the effects of inflammatory and profibrotic cytokine gene expression.
Conclusions.
We have provided further evidence that, despite differences in their
site of inhibition of the RAS, ACEI and AT1RA have
equivalent renal protective effects in 5/6 nephrectomized rats.
Furthermore, in the context of RAS inhibition, the levels of both blood
pressure and urinary protein excretion rates serve as major and
independent determinants of glomerulosclerosis. In addition, the
incomplete suppression of inflammatory and profibrotic gene expression
observed when ACEI or AT1RA treatment is started after the
onset of renal injury may contribute to the slow progression of CRD
during RAS inhibition in this model. Although prospective clinical
trials are required to confirm these findings in humans, it would seem reasonable to conclude that normalization of blood pressure and maximal
reduction of proteinuria should be important therapeutic goals in
clinical strategies aiming to achieve renal protection with RAS
inhibitors. Further studies are required to evaluate whether additional
therapy targeting the effects of inflammatory and profibrotic cytokine
gene expression may further slow the rate of CRD progression.
 |
ACKNOWLEDGEMENTS |
This work was supported by funds from Takeda Chemical Industries
and International Society of Nephrology fellowships (to M. W. Taal
and T. Jiang).
 |
FOOTNOTES |
Present address of G. M. Chertow: Div. of Nephrology,
Univ. of California, San Francisco, 672 Health Sciences East, Box 0532, San Francisco, CA 94143.
Address for reprint requests and other correspondence: M. W. Taal,
Div. of Nephrology, Univ. of California, San Francisco, 672 Health
Sciences East, Box 0532, San Francisco, CA 94143 (E-mail: mtaal{at}rics.bwh.harvard.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 20 March 2000; accepted in final form 24 October 2000.
 |
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