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Regular Articles |
From the Renal and Vascular Laboratory,*
Fundación
Jiménez Díaz, Autonoma University, Madrid, Spain; the
Departments of Internal Medicine
and
Pathology,
Hospital Clinico, Complutense
University, Madrid, Spain; and the Discovery Research
Laboratory,
Tanabe Seiyaku Company, Limited,
Osaka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B and AP-1.
Unexpectedly, AT1(-/-) had a higher interstitial infiltration
than WT. The administration of the angiotensin-converting enzyme
inhibitor quinapril to WT caused a marked improvement in proteinuria
and renal lesions, resembling that seen in untreated
AT1(-/-). However, the interstitial infiltration persisted in
AT1(-/-) when treated with quinapril. Because ET-1 may participate in
the recruitment of mononuclear cells, we also studied the
implication of this peptide. AT1(-/-) had a significantly higher ET-1
expression in tubular epithelial cells than WT. The administration of
the dual ETA/ETB antagonist bosentan to AT1(-/-) considerably reduced
the interstitial infiltrates. Bosentan also exerted a beneficial effect
on proteinuria, renal lesions, and mortality in WT.
These data show that in overload nephropathy, proteinuria and
renal lesions are, to a large extent, AngII-dependent.
The up-regulation of ET-1 in tubular epithelial cells in
AT1(-/-), associated with interstitial infiltrates,
suggests that the combination of drugs interfering with both
vasopeptides may be of therapeutic interest in renal diseases with
severe proteinuria and tubulointerstitial damage.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The prognosis of chronic renal injury, regardless its etiology, is
closely correlated to the severity of tubulointerstitial
damage.12,13
Although persistent proteinuria can induce
tubulointerstitial fibrosis,14-16
underlying mechanisms
still remain unclear. Previous studies from our group have shown that
RAS was activated in tubular cells in animals with persistent
proteinuria.17
Furthermore, ACE inhibitors exert
beneficial effects on the tubulointerstitial damage in this disease and
these effects were attributed, in part, to an attenuation of the
nuclear factor (NF)-B activation.18
Protein overload
nephropathy is considered an appropriate experimental model to approach
the relationship between proteinuria and interstitial damage. The
bovine serum albumin (BSA) overload model is frequently used because it
is highly reproducible and can induce heterologous as well as
autologous proteinuria.16,19-21
In the kidney, AngII exerts its biological effects mainly through AT1.22 In rodents, AT1 exists in two isoforms, AT1A and AT1B, encoded by two different genes. The murine AT1A is the predominantly expressed isoform in most tissues.23 Therefore, the AT1AR-deficient mouse strain [AT1(-/-)] is a powerful model to analyze the effect of AngII blockade.24 In the present study, we further investigated the contribution of RAS in the pathogenesis of tubulointerstitial damage by persistent proteinuria, using the BSA-overload model in AT1(-/-). Because altered bradykinin synthesis and its metabolism,25,26 as well as AT2,27,28 may also be involved in the mechanisms of proteinuria and interstitial cell infiltration, a group of animals was treated with ACE inhibitors. Accumulated evidence has shown that ET-1 also participates in the progression of renal diseases, with or without proteinuria.18,20,29,30 Moreover, a recent study in rats harboring human renin and angiotensinogen genes suggests that ET-1 might mediate inflammatory processes in AngII-induced tissue damage.31 In this regard, we also examined the potential role of ET-1 in proteinuric AT1(-/-) and the potential interrelationship between AngII and ET-1 in the tubulointerstitial damage. The data presented here further extend the implication of both peptides in the pathogenesis of interstitial injury because of proteinuria and support the idea that the combination of drugs modulating both peptides may be useful in nephropathies with persistent proteinuria.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used AT1(-/-) that were generated with a germline chimera derived from TT2 embryonic stem cells with a targeted mutation of the AT1 A gene as previously described.24 AT1(-/-) were back-crossed for more than six generations with C57BL/6 mice.32 As their wild-type littermates (WT), C57BL/6 mice were purchased from Harlan Interfauna Ibérica, S.A. (Barcelona, Spain). Only female mice ages 8 to 10 weeks and weighing 18 to 23 g were used.
Experimental Designs
To decide the experimental condition for murine protein-overload nephropathy, WT weighing 20 g were intraperitoneally injected with four different daily doses (0.1, 0.2, 0.4, 1.0 g) of low endotoxin BSA (Sigma, St. Louis, MO) and followed until day 7. According to their effects on the outcome (see Results), protocols with 0.2 and 0.4 g of BSA daily were used as moderate and severe overload nephropathy models, respectively. Groups of AT1(-/-) and WT, injected with saline, were used as controls in all models.
Groups of mice were sacrificed on days 7, 14, and 28 (moderate) and days 7 and 14 (severe overload nephropathy). Four to 16 mice (BSA-injected WT, AT1(-/-), and their controls) were included at each time point. Urinary protein was determined every day by Knight’s method, as previously described.33 Kidneys of all animals were perfused with cold saline and removed under general anesthesia.
To assess the role of AngII or ET-1 in this disease, we treated animals with moderate and severe nephropathy with the ACE inhibitor quinapril (as powdered hydrochloride salt; Parke-Davis, Barcelona, Spain) or with the dual endothelin type A and B receptor (ETA/B) antagonist bosentan (Ro 47-0203; 4-tert-butyl-N- [6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2'-bipyrimidin-4-yl]-benzene-sulfonamide; Hoffmann-La Roche Ltd., Basel, Switzerland) or their combination. Quinapril (16 mg/kg in distilled water) and bosentan (100 mg/kg in a dissolution of 5% arabic gum)18 were given daily by gastric gavage with a cannula from 24 hours before first BSA injection until day 13.
Renal Histopathological Studies
Paraffin-embedded sections (4-µm thick) were prepared and stained with hematoxylin and eosin and Masson’s trichrome for light microscopy. Semiquantification of morphological lesions were evaluated in regard to glomerular lesions (number of glomeruli with hypercellularity or matrix expansion/30 glomeruli scored 0 to 3), tubular lesions (number of tubuli with dilatation or atrophy/x100 high-power fields scored 0 to 4), protein casts (number of casts/x100 high-power fields scored 0 to 4), and interstitial infiltration (number of focus/kidney scored 0 to 4) by three different pathologists in a blind manner.
For the immunohistochemistry of ET-1,29 kidneys were fixed in 4% paraformaldehyde and embedded in paraffin, and 4-µm thick sections were mounted on poly-L-lysine-coated slides. After deparaffinization with graded concentrations of xylene and ethanol, they were quenched in methanol containing 3% H2O2 at 25°C for 30 minutes to block the activity of endogenous peroxidase. These slides were washed and incubated in trypsin (0.1% trypsin CaCl2 wt/v) to expose antigenic sites. They were subsequently incubated with 5% normal swine serum for 30 minutes at room temperature to reduce nonspecific background staining and then incubated overnight at 4°C with rabbit polyclonal anti-ET-1 antibody (Peninsula Laboratories Europe Ltd., Merseyside, UK). Control slides were treated with diluted normal rabbit serum. After being washed, the sections were incubated with biotinylated swine anti-rabbit IgG (DAKO A/S, Glostrup, Denmark), washed again, and incubated with avidin-biotin-peroxidase complex (DAKO A/S) for 30 minutes. The sites of peroxidase activity were visualized with 0.05% 3,3'-diaminobenzidine (DAKO A/S) in 0.01% H2O2 for 10 minutes. Sections were counterstained with Mayer’s hematoxylin (Sigma, St. Louis, MO) for 2 minutes and coverslipped.
The intensity of ET-1 staining was evaluated by a semiautomatic image analysis system with Microimage version 3.0 for Windows (Olympus, Japan). We quantitatively analyzed ET-1 expression in WT and AT1(-/-) by measuring the integrated optical density (OD) of tubular cells and connective perivascular tissue. OD is a logarithmic function of the light transmitted through the stained section. This formula assumes an exponential decay of light inside the transmitting materials, as follows: OD(x, y) = -log [(intensity(x, y) - black)/(incident - black)]. Intensity(x, y) is the intensity at pixel(x, y), black is the intensity generated when no light goes through the material, and incident is the intensity of the incident light. The system was calibrated in a way that the OD of the negative control was 0.
RNA Extraction and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Pieces of renal cortex were homogenized, and total RNA was
obtained by the acid guanidinium-phenol-chloroform
method.21
One µg of RNA was reverse-transcribed and then
amplified with a commercial kit (Promega, Buckinghamshire, UK), with
the use of 0.5 µCi [
32P]dCTP (3000
Ci/mmol, Amersham) and 20 pmol of specific primers for rat transforming
growth factor (TGF)-ß1 (sense: 5'-AATACGTCAGACATTCGGGAAGCA-3';
antisense: 5'-GTCAATGTACAGCTGCCGTACACA-3'; fragment: 498 bp), rat/mouse
fibronectin (FN) (sense: 5'-TGCCACTGTTCTCCTACGTG-3'; antisense:
5'-ATGCTTTGACCCT-TACACCG-3'; fragment: 312 bp), mouse tumor necrosis
factor (TNF)- (sense: 5'-GCGACGTGGAA-CTGGCAGAAG-3'; antisense:
5'-GGTACAACCCAT-CGGCTGGCA-3'; fragment: 384 bp), and mouse prepro ET-1
(sense: 5'-TGATCTTCTCTCTGCTGTT-3'; antisense:
5'-TTTAAGCTTTTCTGCATGGT-3'; fragment: 408 bp). The amplifications were
performed with annealing temperatures of 62°C (TGF-ß), 60°C (FN),
63°C (TNF-), or 61°C (prepro ET-1). The optimum number of
amplification cycles used for semi quantitative RT-PCR (25, 25, 31, and
32, respectively) was chosen on the basis of pilot experiments (data
not shown). The expression of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was used as internal control. Aliquots of each reaction were
run on 4% acrylamide-bisacrylamide gels. The gels were dried and
exposed to X-OMAT AS films (Eastman Kodak Company, Rochester, NY).
Autoradiograms were quantified by scanning densitometry (Molecular
Dynamics). The density of each gene was compared after the individual
correction by density of GAPDH.
Extraction of Nuclear Proteins and Electrophoretic Mobility Shift Assay
Nuclear extracts were obtained as previously described18,28 and the activity of transcription factors was evaluated by electrophoretic mobility shift assay. Briefly, frozen kidney pieces were pulverized in a metallic chamber and resuspended in a cold extraction buffer [20 mmol/L Hepes-NaOH, pH 7.6, 20% (v/v) glycerol, 0.35 mol/L NaCl, 5 mmol/L MgCl2, 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 mmol/L dithiothreitol, 0.5 mmol/L phenylmethyl sulfonyl fluoride, 1 µg/ml pepstatin A]. The homogenate was vigorously shaken and the insoluble materials precipitated by centrifugation at 40,000 x g for 30 minutes at 4°C. Supernatants were dialyzed overnight against a binding buffer containing 20 mmol/L Hepes-NaOH (pH 7.6), 20% (v/v) glycerol, 0.1 mmol/L NaCl, 5 mmol/L MgCl2, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol, and 0.5 mmol/L phenylmethyl sulfonyl fluoride. These dialysates were cleared by centrifugation at 10,000 x g for 15 minutes at 4°C and stored in aliquots at -80°C until use. Protein concentration was quantified by the bicinchoninic acid method (Bio-Rad Laboratories, Richmond, CA).
NF-B and AP-1 consensus oligonucleotides
(5'-AGTTGAGGGGACTTT-CCCAGGC-3' and 5'-CGCTTGATGAGTCAGCCGGAA-3',
respectively) were [32P]-end-labeled by
incubation for 10 minutes at 37°C with 10 U T4 polynucleotide kinase
(Promega) in a reaction containing 10 µCi of
[
-32P]ATP (3000 Ci/mmol; Amersham, Arlington
Heights, IL), 70 mmol/L Tris-HCl, 10 mmol/L
MgCl2, and 5 mmol/L dithiothreitol. The reaction
was stopped by the addition of EDTA to a final concentration of 0.05
mol/L. Nuclear proteins (20 µg NF-B and 30 µg AP-1) were
equilibrated for 10 minutes in a binding buffer containing 4%
glycerol, 1 mmol/L MgCl2, 0.5 mmol/L EDTA, 0.5
mmol/L dithiothreitol, 50 mmol/L NaCl, 10 mmol/L Tris-HCl, pH 7.5, and
50 µg/ml poly (dI-dC). When competition assays were performed, the
cold probe was added to this buffer 10 minutes before the addition of
the labeled probe. Labeled probe (0.035 pmol) was added to the reaction
and incubated for 20 minutes at room temperature. The reaction was
stopped by the addition of gel-loading buffer (250 mmol/L Tris-HCl,
0.2% bromophenol blue, 0.2% xylene cyanol, and 40% glycerol) and run
on a nondenaturing, 4% acrylamide gel at 100 V at room temperature in
89 mmol/L Tris-borate, 2 mmol/L EDTA, pH 8.0 (TBE).18,21
The gel was dried and exposed to X-OMAT AS films (Eastman Kodak
Company). Autoradiograms were quantified by scanning densitometry
(Molecular Dynamics).
Statistical Analysis
Results are expressed as mean ± SD. Comparisons between groups were made using unpaired Student’s t-test. For statistical analysis of mortality (survival rate), chi-square test using EPI INFO Ver. 6.04 (Centers for Disease Control, Atlanta, GA) was used. Differences were considered as significant if the P value was <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The protein overload nephropathy model is well established in
rats, but not in mice. Initially, we tested the appropriate amounts of
BSA for the murine protein overload model. We used four different daily
intraperitoneal doses of BSA and followed urinary protein level and
mortality until day 7 (Figure 1)
. Mice
weighing 20 g injected with 1 g of BSA daily showed severe
proteinuria, and all animals died before day 7. In contrast, 0.1 g
of BSA daily was not enough to induce pathological proteinuria.
Therefore, we finally used 0.2 and 0.4 g of BSA daily to elicit
the moderate and severe overload nephropathy models, respectively.
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Moderate Overload Nephropathy
In AT1(-/-) and their WT littermates injected with 0.2 g of
BSA, we followed the kinetics of proteinuria for 4 weeks (Figure 2a)
. Peak proteinuria in AT1(-/-) appeared
earlier than in WT, although there was no significant difference
between them. In AT1(-/-) proteinuria decreased after day 7, whereas
in WT it was still elevated until day 14. Both groups did however
reveal moderate proteinuria after 3 weeks.
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Both groups of animals injected with 0.4 g of BSA daily
showed threefold to fourfold higher proteinuria peaks than those of
moderate models, but their kinetics were basically similar (Figure 2b)
.
AT1(-/-) showed high proteinuria from day 1, returning to the normal
limits at approximately day 7. By contrast, severe proteinuria was
persistent in WT for several days with an elevated mortality at
approximately day 14 (69% animals). AT1(-/-) had a significantly
less mortality (22% animals, P < 0.05) at that time.
AT1(-/-) Have Less Renal Lesions, TGF-ß, and Matrix mRNA Expression than WT
In both groups of animals with moderate and severe overload
nephropathy, we evaluated the renal damage at days 7 and 14. Only data
on the severe model are depicted in Figure 3
.
Morphological lesions were significantly less marked in AT1(-/-) than
in WT only at day 14 (Figure 3a)
. Differences in the moderate
nephropathy were less clear between AT1(-/-) and WT (not shown).
Semiquantitative RT-PCR also showed that TGF-ß and FN mRNA expression
was significantly attenuated in AT1(-/-) in relation to WT at day 14
(Figure 3, b and c)
. At day 7, FN expression in AT1(-/-) was
significantly attenuated in both models (moderate model: 1.1 ±
0.2-fold versus 3.4 ± 1.2-fold increase,
n = 3, P < 0.05; severe model; Figure 3c
). On the other hand, we found a significant diminution of TGF-ß
expression in AT1(-/-) at day 7 in moderate overload nephropathy
(1.4 ± 0.1-fold versus 1.8 ± 0.3-fold increase,
n = 4, P < 0.05), but not in severe
nephropathy (Figure 3b)
. As it is well known, AngII is a strong inducer
of TGF-ß. To search for another potential candidate that could
explain the high expression of TGF-ß at day 7 in AT1(-/-), we
examined the gene expression of TNF- one of the most important
inflammatory cytokines able to elicit TGF-ß
expression.34,35
We noted that TNF- expression in
AT1(-/-) with severe nephropathy at day 7 increased as in WT in
relation to controls (3.5 ± 0.5-fold versus 3.8
± 1.4-fold increase, n = 4 to 5, P =
0.76).
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B and AP-1 between AT1(-/-) and WT
Several groups have previously demonstrated that overload
proteinuria induces NF-B activation in tubular epithelial cells both
in vivo and in vitro.18,31,36,37
To
further approach the underlying mechanisms by which AngII participates
at transcriptional levels, we examined NF-B and AP-1 activation in
both groups of animals with severe overload nephropathy.
Electrophoretic mobility shift assay showed that NF-B in AT1(-/-)
was activated like that in WT at day 7, but significantly attenuated at
day 14 (Figure 4)
. NF-B activation was
correlated with the urinary protein levels. Although AT1(-/-)
revealed high proteinuria before day 7, AP-1 activation in those mice
was significantly attenuated in relation to WT along the disease course
(Figure 4)
.
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To further confirm whether the attenuation of overload nephropathy
in AT1(-/-) was because of the absence of AT1 stimulation, a group of
animals was treated with the ACE inhibitor quinapril. AT1(-/-) with
moderate overload nephropathy showed, regardless of quinapril, the same
kinetics of urinary protein excretion (Figure 5a)
, but the early peak of proteinuria in
AT1(-/-) with severe overload nephropathy was delayed by quinapril
(Figure 5b)
. WT with moderate or severe overload nephropathy and
treated with quinapril had a significant reduction in proteinuria with
a pattern very similar to that of untreated AT1(-/-). Mortality in
treated WT with severe nephropathy was also decreased (20%
versus 69% in untreated WT; n = 5 to 13,
P = 0.117).
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Because we have previously demonstrated that ET-1 also
participates in this disease in rats,18,29
we used the
dual ETA/ETB antagonist bosentan in this murine model. In WT with
severe overload nephropathy, bosentan significantly decreased severe
proteinuria (Figure 6a)
, renal lesions
(6.3 ± 1.0 versus 11.7 ± 2.3, n
= 3 to 4, P < 0.01), and improved survival rate
(Figure 6b)
. Although we could not find statistical significance, WT
treated with the combination of bosentan and quinapril showed an
additional improvement in relation to those treated only with bosentan
in proteinuria (Figure 6a)
, renal lesions (5.5 ± 0.6,
n = 4, versus WT, P <
0.01), and survival rate (Figure 6b)
. In WT with moderate nephropathy,
both bosentan and the combined treatment with quinapril also had a
certain beneficial effect on proteinuria (data not shown). However, in
AT1(-/-) treated only with bosentan, the improvement in proteinuria
and survival rate at day14 was less marked (data not shown).
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AT1(-/-) were protected from persistent proteinuria and
glomerular and tubular lesions, but in moderate nephropathy they showed
a significantly higher interstitial infiltration than WT at day 14,
mainly located around small vessels in cortex (Figure 7)
. In severe nephropathy, we failed to find
a significantly marked cell infiltration in AT1(-/-), because of that
WT also showed an important infiltration.
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. We also examined the effect of quinapril on the
interstitial infiltration, but could not find any additional protective
effect, as it was the case in bosentan-treated mice (data not shown). AT1(-/-) Show a Higher ET-1 Protein Expression than WT in Tubular Epithelial Cells
To approach a potential explanation for the cell infiltration
observed in AT1(-/-), we examined ET-1 by immunohistochemistry and
gene expression. AT1(-/-) with moderate overload nephropathy showed
at day14 higher ET-1 expression in tubular epithelial cells and in the
connective tissue around the vessels than WT (146 ± 98
versus 17 ± 23 OD, n = 5,
*P < 0.01) (Figure 8)
. We
also examined the prepro ET-1 mRNA expression in whole kidney from both
animals. Semiquantification studies revealed that AT1(-/-) had a
higher expression than WT, but without statistical significance (at day
7: 1.4 ± 0.1-fold versus 1.0 ± 0.4-fold
increase, n = 3, P = 0.14; at day 14:
1.8 ± 0.3-fold versus 1.3 ± 0.4-fold,
n = 4, P = 0.09). However, there were
no significant differences between AT1(-/-) and WT with severe
overload nephropathy.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BSA-induced overload nephropathy has already been established in rats, but not in mice. Only one study on chronic murine model has been recently published.38 Therefore, we initially examined the experimental conditions to have a reproducible model in mice. Although the adequate range of daily BSA to induce intense proteinuria and renal damage was very narrow, we were able to define two different pictures of overload nephropathy that, in accordance with the intensity, were named moderate or severe. The obtained model in mice resembles to that seen in rats with regards to proteinuria, and glomerular and tubulointerstitial damage. Based on these findings, we induced this model in AT1(-/-) to study the contribution of RAS to the pathogenesis of tubulointerstitial damage because of persistent proteinuria. In addition, we compared both models (moderate and severe) to clarify whether the involved mechanisms are affected by the intensity of proteinuria, which is an important clinical parameter for the efficacy of RAS blockade.5
Overload Nephropathy Is Attenuated in AT1(-/-)
The present study shows that renal injury and mortality at day 14 in overload nephropathy were significantly decreased in AT1(-/-). These data are consistent with the hypothesis that RAS participates in the pathogenesis of tubulointerstitial injury because of persistent proteinuria.17,18
Because the intensity of the early peak of proteinuria was the same in
WT and AT1(-/-), with both moderate and severe overload nephropathy,
initial damage in glomerular epithelial cells should be the same in
both types of mice.19
Consequently, tubular epithelial
cells must be exposed to considerable amounts of urinary proteins
before day 7. It is well known that persistent proteinuria is a key
factor to accelerate the tubulointerstitial damage in human and
experimental renal diseases.14,15
We have previously shown
that persistent proteinuria elicited RAS activation in tubular
epithelial cells.17
Because AngII has been considered a
potent inflammatory mediator,11
the increased local AngII
generation might directly contribute to the disease progression. As
AngII causes an augmentation in TGF-ß and FN, it was not surprising
that AT1(-/-) with moderate overload nephropathy had significantly
less TGF-ß and FN mRNA expression. The importance of AngII in the
synthesis of TGF-ß is supported by several studies showing its marked
reduction (
45%) in animals with various models of renal injury
treated with ACE inhibitors or AT1 antagonists.39,40
However, in AT1(-/-) with severe overload nephropathy, TGF-ß was
only partially decreased, suggesting that other inflammatory mediators,
besides AngII, could be implicated. In fact, animals at day 7 showed
high TNF- mRNA expression in relation to controls.34,35
Moreover, NF-B, a regulator of TNF-,41
was similarly
activated at day 7, but attenuated at day 14 concomitantly with the
absence of persistent proteinuria, therefore suggesting that
proteinuria may directly cause NF-B activation and subsequent
inflammation in an AT1 unrelated manner. By contrast, AT1(-/-) had
significantly less AP-1 along the disease, even in the severe
nephropathy, suggesting that the activation of this transcription
factor is not linked to persistent proteinuria but dependent on the
stimulation of AT1. In fact, in vascular smooth muscle cells, AngII
activates AP-1 through AT1 by a signaling mechanism dependent on
protein kinase C and protein tyrosine kinase.42
Also,
stretch activation of mesangial cells causes FN up-regulation that
requires AP-1 activation.43
In addition, AngII-induced FN
mRNA expression in fibroblasts is regulated by a transcriptional
control on AP-1 site and a posttranscriptional control based on mRNA
stabilization by TGF-ß.44
In this context, the reduction
of FN expression associated with the attenuation of AP-1, but not
NF-B, activation in AT1(-/-) is compatible with these data.
Contrary to that observed in the early stage of the disease, after day
7 AT1(-/-) showed significantly less proteinuria in both moderate and
severe nephropathy, and consequently less tubulointerstitial damage,
that coincided with a significant attenuation of NF-B activation. It
is well known that RAS blockade by ACE inhibitors or AT1 antagonists
decreases proteinuria in human and experimental renal
diseases.1-7
Although the anti-proteinuric effect of ACE
inhibitors was related to the enhancement of kinin
activity,45,46
other reports have shown that these effects
were independent.47
Alternatively, it has also been shown
that ACE inhibitors and AT1 antagonists were equally effective in
reducing proteinuria.48
In our models, urinary protein
kinetics was the same in WT receiving ACE inhibitors and AT1(-/-),
suggesting that the anti-proteinuric action after day 7 was mainly
dependent on AT1 blockade. In fact, the anti-proteinuric effect of RAS
blockade has been attributed mostly to the interference with glomerular
hemodynamics and intrinsic permeability of the glomerular
membrane.40
Interestingly, AT1(-/-) had the proteinuria
peak earlier than WT, especially in the severe overload model,
indicating that AngII may functionally participate in the regulation of
glomerular hemodynamics in the early phase of this disease. Studies in
rats with the amino-acid infusion model demonstrated that AngII
regulates glomerular hemodynamics through the NO
synthesis.49
A similar mechanism might be involved in the
early phase of protein overloading. Because AT1(-/-), with or without
quinapril treatment, showed the same kinetics of urinary proteins,
these functional alterations might be mainly because of an AT1-mediated
mechanism. However, we found a different kinetics in treated and
untreated animals with severe overload nephropathy, suggesting that
kinins may have a role in this alteration.
Interstitial Cell Infiltration Is Higher in AT1(-/-) than in WT with Overload Nephropathy
By semiquantitative evaluation of glomerular and tubular lesions,
protein casts and interstitial infiltration, we noted an attenuation of
renal damage in AT1(-/-). However, taking into account the
chemotactic properties of AngII via AT1,50
it was
surprising to note that AT1(-/-) still have a marked interstitial
cell infiltration mainly detected around the small vessels and in a
focal manner. Those higher infiltrations in AT1(-/-) were conspicuous
in moderate nephropathy, because WT with severe nephropathy finally
showed severe interstitial infiltration and thus higher scores than
AT1(-/-). In this sense, quinapril-treated rats in chronic phase also
showed a certain cell infiltration although there was attenuation in
the total renal lesion score. Recent data suggest that AT2 can also be
implicated in the recruitment of mononuclear cells.28,51
In fact, this receptor has also been involved in NF-B
activation28
and in the expression of some chemokines such
as RANTES.51
The absence of a marked reduction of the
interstitial cells in quinapril-treated AT1(-/-), could be because of
the inability of those drugs to completely inhibit the angiotensin II
generation. In this regard, further experiments are needed (eg, the
treatment with AT2 antagonist) to solve this issue. In addition, other
molecules and intracellular pathways could be implicated in the
mononuclear cell infiltration observed in overload nephropathy.
Beneficial Effects of ETA/ETB Blockade
In several models of renal damage characterized by intense
proteinuria a number of studies have revealed overexpression of ET-1,
mainly located in tubular epithelial cells and, to a lesser extent, in
glomeruli.20,48
ET-1 may induce NF-B activation in
tubular epithelial cells via ETA and ETB receptors.18
The
present study shows that in WT with severe overload nephropathy, ET-1
blockade with bosentan exerted significant beneficial effects on
proteinuria, renal lesions, and mortality. Although ET-1 has a
proinflammatory effect,48
findings in double-transgenic
rats (dTGR) harboring human renin and angiotensinogen
genes31
suggest that ET-1 may be secondarily activated by
AngII. In fact, bosentan significantly reduced AngII-induced NF-B
activation, adhesion molecule expression, and macrophage infiltration
in a blood pressure-independent manner in these animals.31
Moreover, several studies on angiotensin-mediated
hypertension52
and hypertrophy53
also suggest
the existence of an interrelationship between AngII and ET-1 (Figure 9
, pathway 2).
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, pathway 3). AT1(-/-)
did indeed show a higher ET-1 expression in tubular epithelial cells
than WT, despite the absence of persistent proteinuria. In this regard,
it is uncertain if the same occurs in tubular epithelial cells, but
AngII elicits ET-1 synthesis via AT1 in vascular smooth muscle cells
(VSMC).54,55
Furthermore, although we failed to
find statistical significance, WT with quinapril and bosentan showed a
further improvement in proteinuria, renal lesions, and mortality than
those treated with quinapril or bosentan alone. In agreement, the
combined therapy was also superior to each drug alone in rats with
chronic overload nephropathy.18
Those findings further
support the idea that both peptides contribute in parallel to the
pathogenesis of this disease (Figure 9
, pathways 1 and 3) as previously
suggested.56,57
In addition, the fact that proteinuric
AT1(-/-) had ET-1 overexpression and a marked interstitial cell
infiltration postulates their interrelationship (Figure 9
, pathway 2)
and does suggest not only an additive, but also a synergistic benefit
of the combined therapy. On the whole, we have shown that the renal tubulointerstitial damage caused by persistent proteinuria is attenuated in mice strain lacking AT1. The up-regulation of ET-1, mainly in tubular cells, as well as the major beneficial effect of the combined therapy with ACE inhibitors and ETA/ETB antagonists, suggest the implication of AngII and ET-1 in the pathogenesis. The present study further indicates that this combination may afford a synergistic therapeutic effect in renal diseases with severe proteinuria and tubulointerstitial damage.
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Acknowledgements |
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Footnotes |
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Supported by the Comunidad Autónoma de Madrid (grants 08.9/0002.1/2000, 08.4/0019.1/2000), the Fondo de Investigación Sanitaria (grants 99/0425, 00/0111), the Ministerio de Educación y Ciencia (grant PM 97/85), EU Concerted Action Grant (BMH 4-CT98-3631, DG 12-SSMI), and the Japan Health Science Foundation (to Y. S.).
This work was partly presented as oral communication in the meeting of American Society of Nephrology (Toronto, Canada, October 2000). O. L. F., N. T., and R.B. are fellows from Fundación Iñigo Álvarez de Toledo, Fundación Conchita Rábago and Fundación Fernández-Cruz, respectively.
Accepted for publication July 23, 2001.
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