Age-related progressive renal fibrosis in rats and its
prevention with ACE inhibitors and taurine
Carmen Iglesias-De La
Cruz1,
Piedad
Ruiz-Torres1,
Raimundo García
del
Moral3,
Manuel
Rodríguez-Puyol1, and
Diego
Rodríguez-Puyol2,4
Departments of 1 Physiology and 2 Medicine,
Alcalá University, Madrid; 3 Department of
Pathology, Granada University, 18012 Granada;
and 4 Nephrology Section, Hospital Príncipe de
Asturias, 28871 Alcalá de Heuares, Madrid, Spain
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ABSTRACT |
Our previous studies
demonstrated an increased reactive oxygen species (ROS) production, as
well as transforming growth factor-
1 (TGF-
1) expression in the
rat kidney with aging. In the present study, we examined the effect of
aging on extracellular matrix (ECM) accumulation and the effects of
treatment with angiotensin-converting enzyme inhibitors (captopril and
lisinopril) and taurine, an antioxidant amino acid. Age-related
increases in types I and IV collagen and fibronectin mRNA expression
were found at 24 and 30 mo of age. In contrast, type III collagen only
increased in 30-mo-old rats. Captopril-, lisinopril-, and
taurine-treated animals showed a statistically significant decrease in
ECM protein expression at both ages. Moreover, treatment with taurine
reduced the TGF-
1 mRNA levels in 24- and 30-mo-old rats by 40%.
Taurine also completely blocked increases in type I and type IV
collagen expression in mesangial cells in response to TGF-
1. Our
results demonstrate a protective role from both converting enzyme
inhibitors and taurine in the age-related progressive renal sclerosis.
In addition, taking into account that taurine is considered as an
antioxidant amino acid, present data suggest a role for ROS in
age-related progressive renal fibrosis, perhaps through
interactions with the TGF-
1 pathway.
transforming growth factor-
1; extracellular matrix proteins; angiotensin-converting enzyme inhibitors; reactive oxygen species; antioxidants
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INTRODUCTION |
FROM A MORPHOLOGICAL POINT of view, aging is
characterized by the development of structural changes, including
progressive renal sclerosis with glomerulosclerosis and interstitial
fibrosis (9, 16, 21). The biochemical nature of these changes has not
been completely defined, and the most detailed approach to the analysis
of this problem was the study from Abrass et al. (1). These authors
demonstrated by immunohistochemical techniques increased accumulation
of different laminin isoforms in the glomerulus, increased fibronectin
content, and increased interstitial accumulation of collagen types I
(COL I) and III (COL III) in aging rats (1). Surprisingly, no
significant changes in collagen type IV (COL IV) expression were
detected (1). Therefore, the results of this study suggest that the
biochemical nature of the aging-related progressive renal sclerosis
differ from other forms of progressive renal disease (1, 8, 14, 31).
However, immunofluorescence techniques may lack the necessary
sensitivity to detect certain changes in the expression of proteins in
renal tissue, and the study by Abrass et al. (1) would need to be
supported by additional, more sensitive analyses.
The mechanisms involved in the development of the morphological changes
associated with aging have not been definitely elucidated. As
transforming growth factor-
1 (TGF-
1) is one of the most relevant cytokines involved in the pathogenesis of some renal diseases characterized by the accumulation of extracellular matrix (ECM) (3, 17,
25, 28), we tested the hypothesis that TGF-
1 is increased in old
rats. We demonstrated a significant increase of this cytokine in the
kidney in 24-mo-old rats (23). Moreover, as in other experimental
conditions (5, 16, 18, 22), angiotensin-converting enzyme inhibitors
(ACEI) prevented the increased TGF-
1 mRNA expression observed with
age (23). However, although it is generally accepted that the changes
in ECM proteins can be a consequence of the changes in TGF-
1 (3, 17,
25, 28), a detailed analysis of this association has not been performed in aging.
Treatment with ACEIs is the most widely used strategy to prevent
progressive renal sclerosis in different pathological conditions, including aging (5, 16, 18, 22, 23). However, other alternatives could
be useful. A close relationship exists between the increased reactive
oxygen species (ROS) synthesis and aging (29). Antioxidant treatment
has been proposed to prevent aging-related general disturbances (20).
In the kidney, the local synthesis of ROS, at least in experimental
animals and cultured cells, seems to increase with age (24), and
antioxidant treatment could also prevent the morphological and
functional aging-related renal changes
The present experiments were designed to address some of the problems
previously mentioned. First, we tried to perform a more detailed
analysis of the changes in the ECM proteins in the renal cortex from
old rats, by studying the changes in the protein content and the mRNA
of these proteins. Second, we demonstrated that ACEIs treatment not
only reduces the level of TGF-
1 mRNA expression but also reduces the
expression of the mRNAs of different ECM proteins. Finally, we tested
the effect of taurine, an amino acid with antioxidant properties (2,
10, 13), on the development of the aging-related accumulation of these proteins.
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MATERIALS AND METHODS |
Experimental design in vivo. Male Fischer 344 rats were fed a
standard laboratory diet (protein 17.2%, fat 2.7%, fiber 3.9%, minerals 4.4%, carbohydrates 59.7%, and calories 3,100 kcal/kg) and
provided with water ad libitum. The maximal lifespan of this strain is
about 36 mo (30), and the mortality in our 30-mo-old animals was 19%.
In the first part of the study (effect of aging), rats were killed at
3, 24, or 30 mo of age (n = 9 at each time point). In the
second part of the study (effect of treatments), we divided 21- and
27-mo-old rats into three groups (7 animals per group). One group was
treated with captopril (100 mg/l in drinking water, ~10 mg/kg per
day) (15), the second group received lisinopril (28 mg/l in drinking
water, ~2.8 mg/kg per day), and the third group was treated with
taurine (2% in drinking water). Treatment supplies were changed every
2 days, and treatments never exceeded 3 mo. Seven days before the end
of the experimental period, blood pressure (BP) was measured by the
tail-cuff method in conscious animals in each group. Urine samples (24 h) were collected and centrifuged to remove contaminants and then
stored a
20°C until analysis for protein content. At death,
rats were anesthetized with ether, and blood samples were taken from
the lower aorta into tubes containing 7.5% EDTA. Plasma was frozen at
20°C until analysis for creatinine content. After perfusion
with isotonic saline, kidney cortex was excised and placed immediately
in solution D (4 M guanidinium isothiocyanate, 25 mM sodium
citrate, pH 7, 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol) for RNA
extraction or in an embedding medium for frozen tissue specimens (OCT
compound; Miles, Elkhart, IN) for protein extraction, and these samples were immediately frozen at
80°C.
A group of control rats was used to test the antioxidant ability
of taurine. For that purpose, 3-mo-old rats received taurine (2% in
drinking water) for 3 mo, and the malonyldialdehyde (MDA) content in
renal cortex was measured as described (24).
Experimental design in vitro. Experiments were performed in
cultured human mesangial cells. Cells were cultured under standard conditions, as previously described (6). In short, portions of
macroscopically normal, cortical tissue were obtained from a human
kidney immediately after nephrectomy for a renal cell carcinoma. The
cortex was cut into slices and washed twice to remove contaminating
blood; then, the material was pushed through 180-µm and 105-µm
stainless steel sieves and washed to obtain isolated glomeruli free
from tubular contamination. Hanks' balanced salt solution (Flow
Laboratories, Woodcock Hill, UK) was used in all the steps of the
glomerular isolation. Glomeruli were then treated with collagenase type
IA (Sigma, St. Louis, MO), plated on plastic culture dishes, and
maintained in RPMI 1640, supplemented with 10% fetal calf serum
(BioWhittaker, Walkersville, MD), L-glutamine (1 mM),
penicillin (0.66 mg/ml), and streptomycin sulfate (60 mg/ml), and
buffered with HEPES and bicarbonate, pH 7.4 (BioWhittaker), in a 5%
CO2 atmosphere at 37°C. Culture media were changed
every 2 days. When cells reached confluence, they were subcultured at a
ratio of 1:4, using the same incubation medium. Cells were used at
passages 4 to 8, and their identity was confirmed by standard methods
(6, 7). Before starting the experiments, cells were maintained in
serum-free medium for 72 h. After this period, incubations were
performed as follows: control cells, cells incubated with TGF-
1 (2 ng/ml, Sigma) for 18 h, and cells treated with taurine (50 mM, Sigma)
for 6 h and then with TGF-
1 (2 ng/ml) for an additional 18 h.
Thereafter, mesangial cells were lysed in solution D.
In some experiments, mesangial cells were incubated with 0.1 µM
hydrogen peroxide for 30 min, in presence or not of 50 mM taurine, and
the MDA cell content was measured.
Northern analysis. Total RNA was isolated by repeated
phenol-chloroform extractions and isopropanol precipitations as
described (4). Total RNA (20 µg/lane) was denatured by heating in
formamide/formaldehyde at 100°C for 3 min and was electrophoresed
through 1% agarose gel with 0.66 mol/l formaldehyde, transferred to
nitrocellulose membrane (Amersham Ibérica, Buckinghamshire, UK)
by capillary blotting, and ultraviolet cross linked. Integrity and
equal loading of RNA samples were assessed by methylene blue staining
of the transferred RNA (26). All hybridizations were carried out in a
rotating drum in a temperature-controlled oven. The membranes were
prehybridized at 42°C for 24 h in 5× SSPE (20× SSPE: 3 M NaCl, 0.2 M NaH2PO4, and 0.02 M EDTA),
5× Denhardt's solution (50× Denhardt's: 1% Ficoll, 1%
polyvinylpyrrolidone, 1% BSA, 50% formamide, and 0.1% SDS), and 0.1 mg/ml denatured salmon sperm DNA. cDNA inserts were separated from
their vectors in low-melt agarose and labeled with 50 µCi
[32P]deoxyadenosine 5'-triphosphate
(3,000 Ci/mmol, Amersham Ibérica) using a radiolabeled system
(Ready to go; Pharmacia Biotech, Piscataway, NJ). The probes used were
mouse
1(IV) collagen (gift from Dr. M. Kurkinen,
Detroit, MI), rat
2(I) collagen (gift from Dr. DW Rowe, Farmington, CT), mouse
1(III)
collagen, mouse fibronectin, and mouse TGF-
1 (gifts from Dr. F. Ziyadeh, Philadelphia, PA), and rat glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). Blots were hybridized for 24 h in the same
buffer as used for prehybridization with 106 cpm/ml probe
at 42°C. The membranes were washed twice for 5 min in 2× SSC
(20× SSC: 3 M NaCl and 0.3 M sodium citrate, pH 7.0) at room
temperature and then 5 min in 2× SSC and 1% SDS at 65°C. Autoradiography was performed with intensifying screens (X-OMAT, Kodak) at
80°C for 24-48 h. Blots were stripped in 1%
SDS, 0.1× SSC for 30 min at 100°C and subsequently hybridized
with a cDNA probe for GAPDH to account for small loading and transfer
variations. The densitometric analysis of the films exposed was
performed with an Apple scanner and appropriate software (NIH Image
from the National Institutes of Health).
Protein extraction and immunoblot analyses. Kidney cortex
pieces in OCT were thawed at room temperature and washed several times
with cold PBS supplemented with 0.2 mM orthovanadate (Sigma) and 1 mM
phenylmethylsulfonyl fluoride (PMSF, Sigma). Subsequently, pieces were
homogenized in 1 ml of lysis buffer (50 mM Tris, pH 7.2, 150 mM NaCl,
1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, and
0.2 mM orthovanadate) with a hand homogenizer. The homogenized sample
was spun (10,000 g for 30 min at 4°C), and the protein
concentration in each lysate was determined spectrophotometrically (19). The extracted proteins from kidney cortex lysate, as well as a
sample of a standard type I collagen (Sigma Chemical), were solubilized
by boiling in SDS loading buffer (30 mM Tris · HCl, 2% SDS, 10% glycerol, 0.004% bromophenol blue, pH 8.8), and then electrophoresed on 5% polyacrylamide gels in duplicate (40 µg/lane). High-molecular-weight markers (Pharmacia Biotech) comprising
53,000-212,000 Da were used as standards. The first gel was
stained with Coomassie blue to assess for equal protein loading. The
proteins of the second gel were transferred to a 0.45-µm pore
nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) by
the semidry method (Bio-Rad Laboratories, Richmond, CA). Filters were
stained with Ponceau S (Sigma) to control for equal transfer. The
nitrocellulose membranes were blocked with 10 ml of TTBS buffer (20 mM
Tris · HCl, 0.9% NaCl, and 0.05% Tween 20) with 3%
BSA overnight at 4°C. The membranes were incubated with the primary
antibody, rabbit anti-rat collagen type I (Promega, Madison, WI),
diluted in the same buffer for 2 h at room temperature with gentle
agitation. Thereafter, the nitrocellulose membrane was washed three
times with TTBS with 3% BSA and incubated for 90 min at room
temperature with an alkaline phosphatase-conjugated goat
anti-rabbit IgG (Fc specific; Promega) diluted 1:10,000 in TTBS with
3% BSA. After three final washes with fresh TTBS, the membrane was
incubated in alkaline phosphatase buffer (10 mM Tris, 100 mM NaCl, and
5 mM MgCl2, pH 9.5). Secondary antibody bound to the
nitrocellulose was detected by incubation with a substrate solution
(Sigma), which consisted of nitroblue tetrazolium (330 µg/ml) and
5-bromo-chloro-3-indolyl phosphate (165 µg/ml). The color development
was stopped after approximately 5 min by washing the membrane with
distilled water.
Statistical analysis. The present experiments were performed on
the basis of a paired design. For this purpose, a control rat was
selected for each experimental rat (in the case of the different ages,
one 3-mo-old rat was assigned to each 24- and 30-mo-old rat; in the
case of the treatments, one 24- or 30-mo-old nontreated rat was
assigned to each treated rat). Every sample of these paired rats was
run in the same analytical procedure. For the statistical analysis of
the Northern Blot experiments, the densitometric values of the
different ECM proteins were corrected for the densitometric values of
GAPDH. In every case, the results shown are the mean ± SE, and they
are frequently expressed as percent of their control values. Since
samples with n < 10 the normality of the distribution of
values is uncertain, nonparametric statistics was used for comparisons.
Friedman's test or Wilcoxon's test was used. P < 0.05 was
considered statistically significant.
 |
RESULTS |
Characteristics of old rats. Table
1 shows that urinary protein excretion and
body weight in Fischer 344 rats increased progressively with aging. In
contrast, no changes were detected in systolic BP or in plasma
creatinine levels in rats of different ages. The mean increases of the
messages for COL IV and fibronectin were ~1.5- and 3.2-fold,
respectively, in kidneys of 24-mo-old rats compared with 3-mo-old rats
(Fig. 1). By 30 mo of age, the mean transcript levels rose further to 2.3-fold for COL IV and 5.8-fold for
fibronectin (Fig. 1). The mRNAs encoding for interstitial collagens
(COL I and COL III) also increased with age. In the case of COL I, the
mean increases were of 1.6-fold and 2.4-fold at 24 and 30 mo,
respectively (Fig. 1). COL III did not increase in 24-mo-old rats, but
it did in 30-mo-old animals (Fig. 1). To assess whether these changes
in the mRNA corresponded to changes in the ECM protein content in renal
cortex, the presence of COL I in rats of different ages was tested by
Western blot. As shown in the Fig. 2, COL I
content increased as a function of age. The densitometric analysis of
this increase, performed in three different series of animals, showed
mean increases of 42% and 91% in 24- and 30-mo-old rats,
respectively.
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Table 1.
Age-dependent and treatment-dependent changes in systolic BP, plasma
creatinine concentration, urine protein excretion, and body weight in
Fischer 344 rats
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Fig. 1.
mRNA expression in kidney cortex of different extracellular
matrix (ECM) proteins during aging. A: Northern blots of
total RNA from kidney cortex of 3-, 24-, and 30-mo-old animals.
B: summary of results at the different ages (collagen type III
data are not included, as mRNA expression of this protein in 3- and
24-mo-old animals was minimal). Blots were probed with cDNA encoding
1(IV) collagen (COL IV, circles), fibronectin (FN,
squares), 2(I) collagen (COL I, triangles),
1(III) collagen (COL III), and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Densitometric
analysis of the mRNA expression of the different matrix proteins was
corrected with their respective GAPDH. Results are expressed as percent
of control rats and represent means ± SE of 9 different animals.
*P < 0.05 vs. 3-mo-old rats for every protein considered.
**P < 0.01 vs. 3 and 24-mo-old rats for every
protein considered.
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Fig. 2.
Representative Western blot for collagen type I (COL I) in kidney
cortex from rats of different ages. A faint single band of 200-220
kDa, representing collagen type I (see lane 1 for positive
control with a collagen type I standard), can be detected in the renal
cortex of 3-mo-old rats (lane 2), and it increased in 24-mo-old
(lane 3) and 30-mo-old (lane 4) rats.
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Response of ECM protein mRNA levels to treatment with ACEIs.
Treatment with captopril did not have any effect on the systolic BP
or plasma creatinine level, but urinary protein excretion was significantly lower than in nontreated animals of the same age (Table 1). Similar results were observed with lisinopril (data not
shown). There appeared to be no harmful effect of the treatments, because total body weight was not affected (Table 1) and none of the
animals died during the study. Captopril significantly reduced the
mRNA levels of COL IV, fibronectin, and COL I in 24-mo-old rats
(Fig. 3), whereas it significantly reduced
the mRNA levels of the four ECM proteins analyzed in 30-mo-old animals
(Fig. 4). The quantitative analysis of
these inhibitions shows that the mean reduction in the mRNA
expression of COL IV and COL I ranged between 40-50% irrespective
of age, whereas the mean inhibition of the fibronectin and COL III mRNA
expression was about 85% (in the case of COL III this inhibition was
only observed at 30 mo) (Figs. 3 and 4). When these experiments were
performed with another ACEI (lisinopril), the results were similar to
those found with captopril. Table 2
summarizes the results of the Northern blots performed for COL IV and
COL I normalized for GAPDH mRNA levels, at 24- and 30-mo-old control
and lisinopril-treated animals.

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Fig. 3.
Effects of captopril on mRNA expression of different ECM proteins in
kidney cortex of 24-mo-old rats. A: Northern blots of total RNA
from kidney cortex of control (lane 1) and captopril-treated
(lane 2) 24-mo-old animals. Blots were probed with cDNA
encoding 1(IV) collagen, 2(I) collagen,
fibronectin, and GAPDH. B: summary of the results of control
24-mo-old rats (open bars) and captopril-treated 24-mo-old rats (solid
bars). Densitometric analysis of the mRNA expression of the different
matrix proteins was corrected with their respective GAPDH. Results are
expressed as percent of control rats and represent means ± SE of 7 different animals for each group (treated and nontreated animals).
*P < 0.05 vs. 24-mo-old control rats.
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Fig. 4.
Effects of captopril on mRNA expression of different ECM proteins in
kidney cortex of 30-mo-old rats. A: Northern blots of total RNA
from kidney cortex of control (lane 1) and captopril-treated
(lane 2) 30-mo-old animals. Blots were probed with cDNA
encoding 1(IV) collagen, fibronectin,
2(I) collagen, 1(III) collagen, and
GAPDH. B: summary of results of control 30-mo-old rats (open
bars) and captopril-treated 30-mo-old rats (solid bars). Densitometric
analysis of mRNA expression of the different matrix proteins was
corrected with their respective GAPDH. Results are expressed as percent
of control rats and represent means ± SE of 7 different animals for
each group (treated and nontreated animals). *P < 0.05 vs.
30-mo-old control rats.
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Table 2.
Effect of treatment with lisinopril on the mRNA expression of
1(IV) collagen and
2(I) collagen in renal cortex of
control and treated 24- and 30-mo-old rats
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Response of ECM protein mRNA levels to treatment with taurine.
As in the case of ACEIs, no changes were detected in the general status, systolic BP, and plasma creatinine concentration in the animals
receiving taurine with respect to control rats (Table 1). However,
urine protein excretion significantly decreased in the taurine-treated
rats with respect to their controls (Table 1). As shown in Figs.
5 and 6, the mRNA expressions of COL IV and COL I were significantly reduced by taurine treatment, both in
24- and 30-mo-old rats. In the case of COL IV, the mean reductions were about 60% and 65%, at 24 and 30 mo, respectively (Figs. 5 and
6). Mean decreases of 54% and 58% were detected, after taurine treatment, in the COL I mRNA expression in both groups of old rats,
respectively (Figs. 5 and 6). Taurine,
administered to control rats for 3 mo, showed a significant antioxidant
effect, as it decreased the MDA content in the kidney cortex (control
rats 41 ± 5 nmol/g of tissue, n = 4; taurine-treated rats 8 ± 3 nmol/g of tissue, n = 4, P < 0.05 vs. control
rats).

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Fig. 5.
Effects of treatment with taurine on mRNA expression of collagen types
IV and I in kidney cortex at 24 mo of age. A: Northern blots of
total RNA from kidney cortex of taurine-treated and control 24-mo-old
animals. Blots were probed with cDNA encoding 1(IV)
collagen, 2(I) collagen, and GAPDH. B: summary
of results of control 24-mo-old rats (open bars) and taurine-treated
24-mo-old rats (solid bars). Densitometric analysis of mRNA expression
of the different matrix proteins was corrected with their respective
GAPDH. Results are expressed as percent of control rats and represent
the means ± SE of 7 different animals for each group (treated and
nontreated animals). *P < 0.05 vs. 24-mo-old control rats.
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Fig. 6.
Effects of treatment with taurine on mRNA expression of collagen types
IV and I in kidney cortex at 30-mo-old of age. A: Northern
blots of total RNA from kidney cortex of taurine-treated and control
30-mo-old animals. Blots were probed with cDNA encoding
1(IV) collagen, 2(I) collagen, and GAPDH.
B: summary of results of control 30-mo-old rats (open bars)
and taurine-treated 30-mo-old rats (solid bars). Densitometric
analysis of mRNA expression of the different matrix proteins was
corrected with their respective GAPDH. Results are expressed as percent
of control rats and represent means ± SE of 7 different animals for
each group (treated and nontreated animals). *P < 0.05 vs.
30-mo-old control rats.
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Mechanisms of the taurine-dependent reduction in the ECM protein
expression. Treatment with taurine significantly reduced the
TGF-
1 mRNA levels in the kidney cortex of aged rats. Figure 7 shows that taurine inhibits the
expression of the TGF-
1 mRNA in 24- and 30-mo-old rats. Moreover,
preincubation of human mesangial cells with taurine prevented the
TGF-
1-induced synthesis of COL IV and fibronectin observed in these
cells (Fig. 8). As in the case of animals,
taurine prevented the hydrogen peroxide-induced increased MDA content
in cultured cells (control 0.24 ± 0.02, H2O2
0.34 ± 3, taurine + H2O2 0.23 ± 2; results are expressed as nmol/106 cells;
n = 4; P < 0.05 for H2O2 vs.
the other groups).

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Fig. 7.
Effects of treatment with taurine on mRNA expression of transforming
growth factor- 1 (TGF- 1) in kidney cortex at 24 and 30 mo of age.
A: Northern blots of total RNA from kidney cortex of
taurine-treated and control 24- and 30-mo-old animals. Blots were
probed with cDNA encoding TGF- 1 and GAPDH. B: summary of
results of control 24- and 30-mo-old rats (open bars) and
taurine-treated 24- and 30-mo-old rats (solid bars). Densitometric
analysis of mRNA expression of the different matrix proteins was
corrected with their respective GAPDH. Results are expressed as percent
of control rats and represent means ± SE of 7 different animals for
each group (treated and nontreated animals). *P < 0.05 vs.
nontreated animals.
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Fig. 8.
Effects of taurine on mRNA expression of collagen type IV and
fibronectin in cultured human mesangial cells. Quiescent cells were
incubated for 6 h with 50 mM taurine, and then 2 ng/ml TGF- 1 was
added to cells for an additional 18-h period. Northern blots are
representative of total RNA (n = 3). Blots were probed with
cDNA encoding 1(IV) collagen and fibronectin. Lane
1, control cells. Lane 2: cells incubated with TGF- 1.
Lane 3, cells incubated with taurine and TGF- 1.
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DISCUSSION |
Together with the report from Abrass et al. (1), the present results
constitute a detailed analysis of the changes in ECM proteins in the
renal cortex from old rats, although we cannot restrict the changes
observed in ECM proteins to the glomerular and tubulointerstitial
regions of the kidney. In our study, two normal components of the
mesangial matrix, COL IV and fibronectin, and two abnormal components,
COL I and COL III, increased with age. The increased expression of the
mRNAs of these proteins was progressive, as it was more advanced in
30-mo-old than in 24-mo-old rats. However, in the case of COL III, the
increased expression of its mRNA was only detected at 30 mo. The
changes in fibronectin, COL I and COL IV were qualitatively comparable
with previous descriptions (1). We detected increased COL IV expression
in the kidney cortex of aging rats, whereas Abrass et al. (1) did not
detect an increase in the protein level by immunohistochemistry. The apparent discrepancy between both works may relate to differences in
the sensitivities of both techniques, to changes in the translation or
stability of the mRNA or to the turnover of the proteins studied.
ACEI treatment prevented the accumulation of the different ECM proteins
studied. These results are comparable to those observed in other
pathological conditions, such as diabetes, experimental nephritis, or
the reduced renal mass model with progressive kidney disease (5, 16,
18, 22). Lisinopril appeared as effective as captopril in preventing
these changes, and a 3-mo treatment was enough to significantly block
the increased mRNA expression of ECM proteins, both in 24- and
30-mo-old rats. We selected two structurally different ACEI to
ascertain that the observed effects were the consequence of the
blockade of the angiotensin II converting enzyme and not the
nonspecific action of a particular molecule.
Considering these results together with the previous description of the
decreased expression of the TGF-
1 mRNA induced by the same treatment
in old rats (23) and taking into account the well-described properties
of this cytokine in the synthesis of ECM matrix proteins (3), the
following hypothesis can be proposed for the development of
aging-related progressive renal sclerosis. The local renin-angiotensin
system could be involved in determining an increase in the local
synthesis of TGF-
1 in renal cortex of aging rats. TGF-
1
stimulates the synthesis of ECM proteins, and it probably also blocks
their degradation (3), thus inducing the development of the
glomerulosclerosis and interstitial fibrosis which characterize the
aging process. In this hypothesis, it must not be forgotten that ACEIs
also increase kinin synthesis, and a role for these metabolites cannot
be ruled out.
The most novel data provided by the present experiments are those
related to taurine. This amino acid has been previously used, in other
experimental models of disease, to prevent tissue fibrosis. Thus, in
pulmonary fibrosis and in streptozotocin-induced diabetes mellitus,
taurine supplements prevented the accumulation of ECM in lung (11) and
kidneys (27). It seems that these effects of taurine are linked to the
antioxidant ability of the amino acid (2, 10, 13). In 24- and 30-mo-old
rats, the prevention of the increased mRNA levels encoding ECM proteins in the renal cortex was comparable to that observed with ACEI treatment, thus providing an alternative therapeutic approach to the
prevention of aging-related progressive renal fibrosis. Moreover, the
results of the taurine studies may also provide information of the
importance of ROS in the genesis of the accumulation of ECM proteins in
aging. If it is accepted that the main biological activity of taurine
in our experimental model is as an antioxidant (2, 10, 13) and that the
local synthesis of ROS seems to increase with aging (24),
then it can be proposed that ROS could trigger the cellular mechanisms
to overexpress the mRNA of ECM proteins.
The mechanisms that mediate the taurine effects were evaluated at two
levels. First, the changes in the TGF-
1 mRNA expression after
taurine treatment were analyzed. As shown in the RESULTS, taurine decreased the expression of the TGF-
1 in old rats. This cytokine increases with age in Wistar (23) and in Fischer 344 rats
(data not shown). Second, the TGF-
1-stimulated synthesis of ECM
proteins in cultured human mesangial cells was evaluated in presence of
taurine: the amino acid prevented the effects of TGF-
1 on COL IV and
fibronectin synthesis in these cells. A recent report stresses the
importance of ROS as mediators of the cellular effects of TGF-
1
(12). Taurine would abrogate the TGF-
1-induced synthesis of ROS, the
preceding step in the development of its cellular actions, thus
blocking ECM accumulation. In consequence, taurine blocks both the mRNA
expression of TGF-
1 as well as the cellular actions of the cytokine,
thus providing a useful tool in the prevention of the ECM protein
overexpression that characterizes aging.
The consequences from both ACEI and taurine treatments were not only
morphological but also functional, as proteinuria decreased in old rats
receiving these treatments. Interestingly, the antiproteinuric effect
of ACEI and taurine was less marked when started later in life,
although their ability for blocking ECM mRNA expressions was
maintained. These findings raise important questions about the
relationships between proteinuria and ECM accumulation, as the
decreased proteinuria can be the cause or the consequence of the ECM
changes, but also about the starting time and duration of the selected
treatments and the role of ECM protein turnover in the genesis of the
detected alterations. Additional experiments are needed to adequately
clarify these problems.
In summary, the present results provide a more detailed analysis
regarding the biochemical composition of the aging-related progressive
renal fibrosis. In addition, they point to taurine as an alternative
tool to ACEI for preventing the morphological changes of the kidneys
from elderly individuals. Finally, they suggest a role for ROS in the
pathophysiology of aging-related progressive renal fibrosis, but direct
experimental confirmation is required.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Fuad N. Ziyadeh for the generous gifts of COL III,
fibronectin, and TGF-
1 cDNA (Renal Electrolyte and Hypertension Division and Penn Center for Molecular Studies of Kidney Diseases, Dept. of Medicine, University of Pennsylvania, Philadelphia, PA). We
also thank Dr. Kurkinen (Wayne State University, Center for Molecular
Biology, Detroit, MI) and Dr. Rowe (Rowe Laboratory, Dept. of
Pediatrics. School of Medicine, University of Connecticut Health
Center, Farmington, CT) for the generous gifts of collagen type IV and
collagen type I cDNA respectively.
 |
FOOTNOTES |
This work was supported by Comision Interministerial de Ciencia y
Tecnologia (CICYT) Grant SAF 930713 and by Fondo de
Investigaciones Sanitarias (FIS) Grant 95/0021-01. C. Iglesias-de
la Cruz is a fellow of the Ministry of Education and Science of Spain.
P. Ruiz-Torres is a postdoctoral fellow of the Ministry of Education
and Science of Spain.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Rodríguez-Puyol, Departamento de Fisiología, Facultad
de Medicina, Campus Universitario, Ctra Barcelona KM 33, 28871 Alcalá de Henares, Madrid, Spain (E-mail:
ffmrp{at}alcala.es).
Received 29 December 1998; accepted in final form 6 August 1999.
 |
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