Influence of nephron mass in development of chronic renal
failure after prolonged warm renal ischemia
Josep M.
Cruzado1,2,
Joan
Torras1,2,
Marta
Riera2,
Immaculada
Herrero2,
Miguel
Hueso1,
Luís
Espinosa1,
Enric
Condom3,
Núria
Lloberas2,
Jordi
Bover1,
Jeroni
Alsina1, and
Josep M.
Grinyó 1,2
1 Nephrology and 3 Pathology Departments, Ciutat
Sanitària i Universitàia de Bellvitge, and
2 Laboratory of Nephrology, Department of Medicine, University
of Barcelona, 08907 L'Hospitalet, Barcelona, Spain
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ABSTRACT |
The present study examined the
long-term consequences of warm renal ischemia (WRI) with or without
renal ablation. Male Sprague-Dawley rats (250-300 g) were
subjected to 60 min of complete WRI by pedicle clamping and then
followed for 52 wk. Animals were organized into four groups: rats in
which both kidneys were subjected to warm ischemia (2WIK); rats with
left WRI and right nephrectomy (1WIK); uninephrectomized rats with a
left nonischemic kidney (1NK); and sham-operated rats (2NK). Additional
animals were studied at 24 h, 7 days, and 16 and 32 wk. In the
first week after WRI, rats from the 2WIK and 1WIK groups displayed a
similar degree of acute renal damage. After recovering from acute renal
failure, 1WIK rats developed progressive and severe proteinuria,
whereas it was mild in the 2WIK group, as well as in the 1NK and 2NK
groups. Only animals from the 1WIK group developed severe chronic renal failure, glomerulosclerosis, interstitial fibrosis, and upregulation of
transforming growth factor-
1 (TGF-
1)
gene, which was associated with increased TGF-
1 protein
expression in tubular epithelial cells, arterioles, and in areas of
mononuclear interstitial cell infiltrate. On the contrary, long-term
renal TGF-
1 expression, function, and histology were
similar in 2WIK and 2NK rats. The present study shows that prolonged
bilateral WRI, when both kidneys are retained in place, induces very
mild long-term renal lesions as opposed to the severe renal scarring
observed when WRI is combined with contralateral nephrectomy.
uninephrectomy; renal fibrosis; transforming growth
factor-
1; apoptosis; glomerulosclerosis
 |
INTRODUCTION |
THERE IS AN
INCREASING DEMAND for kidney allografts to treat patients with
end-stage renal disease. Thus organ shortage creates pressure on renal
transplantation teams, which leads them to accept organs from the
so-called "marginal donors" as well as organs from
non-heart-beating donors (NHBD). In a recent work, Cho and colleagues (8) showed that 1-yr graft survival was not
adversely affected by transplanting kidneys from donors whose hearts
had stopped beating. Our group has recently reported a similar clinical experience using non-heart-beating donors (16), although
we observed very poor 5-yr graft survival when warm ischemia time associated to donor's cardiac arrest was >45 min.
It is well known that ischemia-reperfusion injury induces both
necrosis and apoptosis in tubular epithelial cells (7,
29). Apoptosis is an active process of programmed cell
death associated with several physiological and pathological conditions
(23). After warm renal ischemia, both the generation of
reactive oxygen species at reperfusion (11) and the
polymorphonuclear cells that infiltrate the organ (34) may
induce apoptosis in the kidney. In a recent work, Burns et al.
(7) provided evidence that ischemia-reperfusion injury
promotes apoptosis in clinical renal allografts. Moreover, Matsuno et
al. (22) showed that apoptosis correlated with delayed graft function, which is recognized as an important risk factor for
chronic transplant nephropathy (24). Thus, in association with functional and morphological markers of postischemic acute renal
damage, the evaluation of apoptosis contributes to assessing the extent
of ischemia-reperfusion injury in the kidney.
Considering that nephron mass seems to be a critical long-term
prognostic factor in the progression of chronic renal disease, several
questions have emerged in the clinical transplantation arena
(6), such as, is it possible to improve the poor results obtained with organs from older and marginal donors by transplanting two kidneys to a recipient? And if so, is it possible to expand the
pool of kidney donors by using this strategy? Preliminary results of
double-kidney transplantation from older and marginal donors suggest
that this approach may be a good way to expand donor criteria
acceptance (18, 20). Likewise, we believe
that whether this promising strategy is useful in improving graft
survival in renal transplantation from NHBD with prolonged warm
ischemia should be investigated. Experimental studies in rats have
revealed that, even in the absence of alloresponse, 45 or 60 min of
warm renal ischemia associated with contralateral nephrectomy induce long-term glomerulosclerosis, interstitial fibrosis, and vascular myointimal proliferation (32, 33). Therefore,
to explore the long-term potential benefit of supplying double renal
mass when there is prolonged warm ischemia, we performed 60 min of
bilateral warm renal ischemia in rats and followed these animals for 52 wk. We sought to compare warm ischemia-induced acute renal injury, including apoptosis, in uninephrectomized and nonnephrectomized rats
and, then, the long-term renal functional and pathological consequences
of prolonged warm renal ischemia, depending on whether rats had one or
two injured kidneys.
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MATERIALS AND METHODS |
Experimental design.
Studies were performed in male Sprague-Dawley rats (250-300 g body
wt). Animal care and interventions were conducted in accordance with
the Guidelines of the European Community Committee on Care and Use of
Laboratory Animals and Good Laboratory Practice. Anesthesia was induced
with ketamine (75 mg/kg im), atropine (0.05 mg/kg im), and diazepam (5 mg/kg im). For surgery, animals were placed on a warming pad to
maintain body temperature constant within physiological range. After
median laparotomy, both renal pedicles were dissected. Prolonged warm
ischemia was induced in the rats by clamping the renal pedicle for 60 min with a vascular nontraumatic clamp. Right nephrectomy was performed
depending on the experimental group. Animals were divided into four
groups, as follows: rats with bilateral warm renal ischemia (2WIK);
rats with left warm renal ischemia and right nephrectomy (1WIK);
uninephrectomized rats with a left nonischemic kidney (1NK); and
sham-operated rats with two nonischemic kidneys (2NK). After recovery,
animals were housed in a room kept at a constant temperature with a
12:12-h dark-light cycle. Rats had free access to tap water and were
fed a standard rodent diet.
Acute experiments were performed to evaluate renal histology 24 h
after renal ischemia (n = 6 in each of the 4 experimental groups). Additional animals were included in the 1WIK
(n = 6) and 2WIK (n = 6) groups to
study the serum creatinine profile and creatinine clearance and to
analyze renal histology 7 days after ischemia.
In chronic experiments, animals were distributed in the following
groups and monitored for 52 wk: 2WIK (n = 11), 1WIK
(n = 12), 1NK (n = 8), and 2NK
(n = 7). Every 4 wk, animals were placed in
metabolic cages to collect 24-h urine samples. At the same intervals,
0.5 ml of blood was obtained from the tail vein, and body weight was
recorded. Additional animals were included in the 1WIK, 2WIK, and 1NK
groups to evaluate renal histology and transforming growth
factor-
1 (TGF-
1) gene expression by
RT-PCR at 16 and 32 wk (n = 5 for group in each time period).
Biochemical data.
Creatinine was determined by a standard autoanalyzer (Echevarne
Laboratories, Barcelona, Spain). Creatinine clearance (ml · min
1 · 100 g body wt
1) was
calculated by standard formula. Proteinuria (mg/24 h) was measured
every 4 wk by the Ponceau method (Bayer Diagnósticos, Madrid, Spain).
Renal functional studies.
At 52 wk animals were subjected to invasive functional studies to
assess glomerular filtration rate (GFR) and renal plasma flow (RPF) and
to measure arterial blood pressure. For this purpose, rats were
anesthetized again with an intramuscular mixture of ketamine-diazepam-atropine and placed on a heating pad to maintain body
temperature within physiological range. Vascular polyethylene catheters
were implanted into the right carotid artery and right jugular vein. An
arterial catheter was used for continuous monitoring of arterial
pressure by means of an electronic pressure transducer (Nihon Kohden)
and for blood sampling. A venous catheter was used for the infusion of
fluid and clearance markers. The abdominal cavity was opened, and the
ureter was cannulated for the collection of urine with polyethylene
tubing. In the case of using two kidneys, both ureters were cannulated,
allowing measurement of the GFR and RPF from both kidneys
separately. A priming load of inulin and
p-aminohippurate (PAH) solution (83 mg inulin, 5 mg PAH) was administered via the right jugular vein. Then, a continuous infusion of
a solution containing 2% inulin and 0.6% PAH was maintained at a
constant rate of 0.5 ml · 100 g body wt
1 · h
1. After an equilibration period of 60 min, urine
collection was started. To determine inulin and PAH clearances, three
periods of 20 min each were established. At the midpoint of each
period, 0.5 ml of arterial blood was obtained. The inulin concentration in plasma and urine was determined by means of the indole-3-acetic acid
(Merck, Darmstadt, Germany) colorimetric assay. The PAH concentration in plasma and urine was measured by the Branson colorimetric method. GFR and RPF were measured as inulin and PAH clearances, respectively, calculated by standard formulas, and the final result provided was the
mean of the three values from each 20-min period. At the end of the
study, kidneys were perfused with a 4°C 0.9% saline solution and
kidney weight was recorded.
Light microscopy.
Two-millimeter-thick kidney coronal sections were fixed in 10% neutral
buffered Formalin and embedded in paraffin. Four-micrometer-thick tissue sections (samples from 16, 32, and 52 wk) were stained with
hematoxilin and eosin (all samples) and the periodic acid-Schiff method. Tubular dilation, tubular cell detachment, tubular cell necrosis, interstitial infiltrate, and interstitial edema were evaluated in samples obtained 24 h and 7 days after surgery. For this purpose a semiquantitative scale graded from 0 to 4+ was used (0 denoted no abnormalities; 1+, changes affecting <25% of the sample;
2+, changes affecting 25-50% of the sample; 3+, changes affecting
50-75% of the sample; and 4+, changes affecting >75% of the
sample). Glomerulosclerosis was assessed by examining all the glomeruli
in samples obtained at 16, 32, and 52 wk and expressed as the
percentage of glomeruli presenting focal or global sclerotic lesions.
In these samples, tubular atrophy and dilation, interstitial fibrosis,
interstitial mononuclear infiltrate, as well as vascular myointimal
proliferation, were also evaluated by a semiquantitative scale graded
from 0 to 3+ (0, no changes; 1+, changes affecting <1/3 of the sample;
2+, changes affecting between 1/3 and 2/3 of the sample; 3+, changes
affecting >2/3 of the sample).
In situ detection and evaluation of apoptosis.
This technique was used to further assess acute (24-h) renal lesions
induced by warm ischemia in uninephrectomized and nonnephrectomized rats. Apoptotic cells were evaluated by the in situ labeling of nuclear
DNA fragmentation (Apoptag S7100 peroxidase kit, Oncor, Gaithersburg,
MD), following the manufacturer's instructions, in 5-µm-thick
deparaffined renal tissue sections, which were then counterstained with
hematoxylin and mounted under a glass coverslip in DPX (DBH, Merck,
Darmstadt, Germany). With the previous work by Nogae et al.
(25) taken into account, the degree of apoptosis 24 h
after renal warm ischemia was evaluated in tubular cells of the outer
medulla and graded from 0 to 3+ (0, negative; 1+, mild; 2+, moderate;
3+, intense).
Determination of renal TGF-
1 messenger RNA
expression by RT-PCR.
A modification of a previously described semiquantitative RT-PCR
protocol (17) was used to analyze TGF-
1
mRNA expression in renal tissue, using
-actin as a housekeeping
gene. Briefly, total kidney RNA was isolated with TriPure isolation
reagent (Boehringer Mannheim, Mannheim, Germany) based on the
Chomczynski and Sacchi method (9). The final RNA pellet
was resuspended in diethylpyrocarbonate-treated water and quantified by
spectrophotometry at 260 nm. All samples showed a 260/280 optical
density ratio of >1.8. For random-primed first-strand cDNA synthesis,
a sample of 1-2 µg total RNA was incubated with 10 U of DNase I
RNase-free (Boehringer Mannheim) and 20 U recombinant RNasin
ribonuclease inhibitor (Promega, Madison, WI) in a total volume of 10.5 ml at 37°C for 30 min. The reaction was stopped by heating at 95°C
for 15 min. Fifty nanograms of random hexamers were then added, and the
mixture was kept for 10 min at 65°C and immediately put on ice. Two
hundred units of SuperScript RT (GIBCO-BRL, Eggenstein, Germany) were
then added, and RT was carried out according to standard
manufacturer's specifications for 50 min at 42°C in a final volume
of 20 µl. The RT reaction was stopped by heating at 95°C for 5 min.
Negative controls without RT were included for all individual samples.
For PCR analysis, nonlooping-nonoverlapping deoxy-oligonucleotide
primers from separate exons were obtained for the analyzed gene. A rat
-actin primer pair was obtained from Clontech (Palo Alto, CA): a
740-bp fragment was amplified by using a 5'-forward primer,
5'-TTGTAACCAACTGGGACGATATGG-3', and a 3'-reverse primer,
5'-GATCTTGATCTTCATGGGTGCTAGG-3'. TGF-
1 primer pairs
were 5'-forward 5'-CTTCAGCTCCACAGAGAAGAACTG-3' and 3'-reverse
5'-CACGATCATGTTGGACAACTGCTCC-3', which amplified a 318-bp fragment
(26). One microliter of RT product was amplified in a
final volume of 50 µl containing 1.25 U of BIOTaq DNA
polymerase (Progenetic, Vista, CA), 67 mM Tris · HCl buffer (pH
8.8), 16 mM (NH4)2 SO4, 0.1% Tween-20, 2.5 mM
MgCl2, and 200 µM of dNTP. The concentration of the
primers was adjusted to avoid product saturation. Thus 0.062 µM
-actin and 0.12 µM TGF-
1 oligonucleotide primers
were used for PCR amplification. To minimize PCR variability, all PCR
reactions were performed in cDNA aliquots, and individual samples were
processed at the same time for each PCR amplification in a GeneAmp PCR
System 9600 (Perkin Elmer-Cetus, Foster City, CA) under the following
conditions: an initial denaturation step at 94°C for 5 min; 35 cycles
at 94, 60, and 72°C for 45 s, 45 s, and 2 min,
respectively, as denaturation, annealing, and extension steps; and a
final extension at 72°C for 7 min. After amplification, 10 µl of
each PCR reaction mixture were electrophorezed through a 1% agarose
gel in 1× TBE buffer (comprising 89 mM Tris base + 89 mM boric
acid + 2 mM EDTA) containing 0.1 µg/ml of ethidium bromide. The gel was photographed with Polaroid 667 film
(Cambridge, MA) under ultraviolet light exposure. Bands were scanned by
laser densitometry (Epson GT-8500) and analyzed by using specific
software (Phoretix 1D Advanced version 3.01). The optical density of
TGF-
1 was obtained in arbitrary units and expressed as a
ratio to
-actin.
Localization of renal TGF-
1 protein expression by
immunohistochemistry.
Paraffin-embedded sections from kidneys harvested at 52 wk were
immunostained by the immunoperoxidase technique (Vectastain ABC kit
anti-rabbit IgG, Vector Laboratories, Burlingame, CA). As primary
antibody, we used a rabbit polyclonal IgG for the epitope of
TGF-
1 corresponding to amino acids 328-352 of the
COOH-terminal region (Santa Cruz Biotechnology, Santa Cruz, CA). This
antibody is specific for TGF-
1 (non-cross-reactive with
TGF-
2 or TGF-
3) in rat, mouse, and human
tissues. Briefly, 4-µm-thick paraffin-embedded sections were cleared
and rehydrated. After washing with PBS, endogenous peroxidases were
inactivated by 3% H2O2. To optimize antigen
expression, samples were boiled for 25 min in pH 6 citrate buffer.
After three 5-min washes in PBS, samples were incubated for 2 h in
20% goat serum to block unspecific antibody binding. Samples were
incubated overnight with the 1:25 diluted anti-TGF-
1 polyclonal antibody. Then, after washing with PBS, anti-rabbit IgG
(1:200) was added and incubated for 1 h at room temperature. Then,
samples were incubated in avidin-biotin complex 1:100 for 1 h at
room temperature, diaminobenzidine revealed, washed in water,
counterstained with hematoxylin, dehydrated, and mounted in DPX (BDH,
Merck). Negative controls were performed by immunostaining matched
serial sections without the addition of the primary
anti-TGF-
1 antibody, and by preincubation with the
commercially available blocking peptide (Santa Cruz Biotechnology).
Semiquantitative evaluation of TGF-
1 stain was graded
from 0 to 3+ (0, negative; 1+, mild; 2+, moderate; 3+, intense).
Statistical analysis.
All data are presented as means ± SE. To compare more than two
groups for quantitative variables, the one-way analysis of variance
followed by the Scheffé's test was used. Data from the two
groups of animals followed for 7 days were compared by the nonparametric Mann-Whitney U-test. Semiquantitative data
from renal histology, apoptosis, immunohistochemistry, and RT-PCR for TGF-
1 were compared by the nonparametric Kruskal-Wallis
test followed by the Connover test. The statistical significance level was defined as P < 0.05.
 |
RESULTS |
Effect of nephron mass on warm ischemia-induced acute renal
failure.
In the acute experiments, rats from the 2WIK and 1WIK groups had a
similar degree of postischemic acute renal failure, as shown by an
elevated serum creatinine level 24 h after surgery (Fig.
1). Seven days postischemia serum
creatinine returned to the normal level in 2WIK rats, whereas it still
remained increased in 1WIK rats (0.56 ± 0.07 vs. 0.82 ± 0.01 mg/dl, P = 0.009). Again, on day 7,
creatinine clearance was significantly lower in the 1WIK than in the
2WIK group (1WIK = 258 ± 18 vs. 2WIK = 342 ± 23 µl · min
1 · 100g body wt
1,
P = 0.01). Histological scores of warm ischemia-induced
renal damage 24 h and 7 days after ischemia (Table
1) were similar and severe in both
groups. As shown in Fig. 2, main
histological findings 24 h after ischemia were cortical tubular
epithelial cell necrosis and apoptosis, most tubular lumens were
occluded by debris, and there was congestion of the outer medulla. The majority of apoptotic cells were in tubules on the inner stripe of the
outer medulla as observed by the in situ labeling of nuclear DNA
fragmentation (Fig. 2). As shown in Table 1, only ischemic kidneys
(1WIK and 2WIK groups) had apoptosis in tubular cells. In accordance
with light microscopy findings, the degree of apoptosis was similar in
the 1WIK and 2WIK groups. On the other hand, 7 days after warm
ischemia, renal histology revealed that tubules were dilated with
flattened epithelium, interstitial infiltrate, and medullar calcinosis,
with such lesions being nearly the same in 1WIK and 2WIK kidneys (Table
1).

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Fig. 1.
Serum creatinine evolution in uninephrectomized (1WIK)
and nonnephrectomized (2WIK) rats in the first week after warm
ischemia. Basal level denotes the serum creatinine in these animals
before manipulation (0.5 ± 0.01 mg/dl).
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Table 1.
Renal histology and Apoptag staining at 24 h and 7 days after
surgery in ischemic uninephrectomized and ischemic nonnephrectomized
rats
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Fig. 2.
Morphological lesions and in situ detection of DNA strand
breaks by Apoptag 24 h after reperfusion in kidneys from 1WIK and
2WIK rats. A: renal tubules with massive epithelial cell
detachment, tubular cells with apoptotic bodies, and intraluminal
debris (hematoxilin-eosin staining, ×400). B: many
Apoptag-labeled nuclei in tubular epithelial cells of the outer medulla
(×200). C: a control kidney section from a 1NK rat obtained
24 h after surgery (×400).
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Influence of nephron mass on long-term renal functional parameters
after recovery from ischemic acute renal failure.
Mortality rate from acute renal failure in animals included in the
long-term follow-up was 2/11 in 2WIK and 3/12 in 1WIK rats. No
additional mortality was observed thoughout followup in any of the four
experimental groups. As early as 8 wk after ischemia, 1WIK rats
developed progressive and severe proteinuria (Fig.
3). In contrast, 2WIK rats showed mild
proteinuria, which was not significantly higher than that observed in
2NK and 1NK rats. Eight weeks after ischemia, creatinine clearance in
the 1WIK group returned to its basal level. Thus within weeks
8 and 40 all four experimental groups had similar
values of creatinine clearance (Fig.
4A). Nevertheless, starting in
week 40, 1WIK rats developed progressive renal insufficiency (Fig. 4, A and B). Invasive functional studies
performed at the end of the study confirmed these findings more
accurately (Fig. 5) by showing that 1WIK
rats had a significantly lower total RPF and GFR than did 2WIK, 1NK,
and 2NK rats. Interestingly, 2WIK rats had RPF and GFR values as good
as those observed in 2NK animals. Moreover, as shown in Table
2, the analysis of the left kidney clearance parameters in all four groups confirmed that renal function was deeply depressed in 1WIK rats and preserved in 2WIK ones. Arterial
blood pressure was within normal range and similar in all four groups
(Table 2), as previously reported in a nephron supply model
(21).

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Fig. 3.
Proteinuria during the 52-wk follow-up. Starting in
week 8, 1WIK rats had significantly higher proteinuria than
other 3 groups (P < 0.05). There are not significant
differences between 2WIK and uninephrectomized-nonischemic (1NK) or
sham-operated (2NK) rats.
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Fig. 4.
Creatinine clearance (A) and serum creatinine
(B) during the 52-wk follow-up. Four weeks after surgery,
1WIK and 1NK rats had a reduction in creatinine clearance, although
serum creatinine remained within normal range. Thereafter, creatinine
clearance returned to its basal value in the 1WIK and 1NK groups.
Starting in week 40, 1WIK rats developed a significant
reduction in creatinine clearance in parallel with a progressive
increase in serum creatinine (P < 0.05 vs. the other
groups). BW, body wt.
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Fig. 5.
Glomerular filtration rate (GFR) and renal plasma flow
(RPF) at 52 wk. 1WIK rats had lower total GFR and RPF than other
groups, whereas 2WIK rats showed GFR and RPF as good as in 2NK
animals.
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Influence of nephron mass on long-term renal histology after
recovery from ischemic acute renal failure.
As shown in Table 3, at 16 wk, kidneys
from the 2WIK group appeared well preserved and had only minimal
interstitial mononuclear infiltrate. In contrast, 1WIK kidneys had
significantly higher glomerulosclerosis and mononuclear interstitial
infiltrate than did kidneys from the 2WIK and 1NK groups. In the 1WIK
group, renal lesions progressed throughout the follow-up. Thus at 32 wk, these animals had significantly higher glomerulosclerosis, tubular
atrophy, interstitial infiltrate, and myointimal proliferation than did 2WIK and 1NK rats. Moreover, at 52 wk, interstitial fibrosis also became evident in 1WIK rats in comparison to the other groups. Noteworthy, in this final period, kidneys from the 2WIK group had
minimal glomerulosclerosis (2.7 ± 1% in 2WIK vs. 31.2 ± 6.8% in 1WIK, P < 0.05), without evidence of
interstitial fibrosis, interstitial infiltrate, or vascular lesions,
although some tubules were atrophied.
Nephron mass effect on long-term renal TGF-
1 mRNA
expression and protein localization after recovery from postischemic
renal failure.
Kidneys from the 1WIK group showed a significant increase in
TGF-
1 mRNA expression throughout the follow-up
(P = 0.02) (Fig. 6).
Conversely, TGF-
1 mRNA in the 2WIK rats remained stable
during the study period (16, 32, and 52 wk). As early as 16 wk after ischemia (before the appearance of severe renal lesions), 1WIK kidneys
had higher TGF-
1 mRNA than did 1NK kidneys. Moreover, TGF-
1 gene expression at 32 and 52 wk was higher in the
1WIK group than in the 2WIK group. Noteworthy, at the end of the study, TGF-
1 mRNA was similar in 2WIK and 2NK kidneys. All four
experimental groups showed positive staining for TGF-
1
in tubular and glomerular cells, but its expression was clearly more
intense in 1WIK than in 2WIK (P = 0.03), 1NK
(P = 0.03), and 2NK (P = 0.02) groups (Table 3). In kidneys from the 1WIK group, TGF-
1 protein
was mainly enhanced in the cytoplasm of tubular cells, renal arterioles (Fig. 7), and in mononuclear interstitial
infiltrates.

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Fig. 6.
mRNA expression of transforming growth
factor- 1 (TGF- 1). The 52-wk value of
TGF- 1 mRNA of the 2NK group is represented by the open
box. Only 1WIK rats showed a significant increase in
TGF- 1 mRNA expression throughout follow-up
(P = 0.02). 2WIK rats had a stable level of
TGF- 1 mRNA, which was similar to that in 2NK rats. O.D.,
optical density.
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Fig. 7.
Immunohistochemistry for TGF- 1 in ischemic
uninephrectomized (A) and ischemic nonnephrectomized
(B) rats. Note the more pronounced and diffuse tubular
staining as well as intense perivascular staining of a renal arteriole
(arrow) in the kidney from the ischemic-uninephrectomized rat
(A). C: a kidney section from a 52-wk follow-up
2NK rat showing a very weak tubular TGF- 1 staining.
Magnification: ×400 in A-C.
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DISCUSSION |
The main finding of our study is that prolonged warm renal
ischemia was insufficient to induce chronic renal damage in the absence
of renal ablation. This is supported by the evidence that 52-wk
renal function and renal histology were similar in two-ischemic kidney
and sham-operated rats, whereas ischemic-uninephrectomized rats
developed severe chronic renal insufficiency, glomerulosclerosis, interstitial fibrosis, and myointimal proliferation.
Animal studies have revealed that severe ischemia and reperfusion
injury, when combined with contralateral nephrectomy, induces long-term
glomerulosclerosis, interstitial fibrosis, and myointimal proliferation
(32, 33). This experimental model is useful for testing protective agents (32) because it resembles
the clinical renal transplantation setting in the absence of
alloresponse, in which only one kidney is grafted. On the other hand,
Azuma et al. (2) demonstrated that in this model, the
chronic renal lesions are attenuated when the nonischemic contralateral
kidney is retained in place. Nevertheless, the long-term consequences of bilateral warm renal ischemia have not been previously investigated.
Ischemia-reperfusion injury is an inflammatory process
(37) governed by adhesion molecules and cytokines
(30), which induce necrosis and apoptosis in tubular
epithelial cells (7, 29). There is evidence
suggesting that ischemia-reperfusion injury causes acute organ
dysfunction and initiates a cascade of events leading to renal scarring
(31). Previous studies analyzed the effect of renal mass
on postischemic acute renal failure. Finn et al. (13),
comparing functional and pathological findings after unilateral warm
renal ischemia in uninephrectomized and nonnephrectomized rats,
found that the presence of a contralateral normally functioning kidney
aggravated the acute renal injury induced by 60 min of warm ischemia.
In fact, the presence of a nonischemic contralateral kidney was
associated with a more severe preglomerular vasoconstriction and a
widespread tubular obstruction in the ischemic kidney. Similar findings
were described in a syngeneic rat renal transplant model when one or
two functioning native kidneys were retained in place
(10). These authors suggested that the detrimental effect
induced by the native kidneys could be mediated either by potentiation
of the severity of ischemic injury or, alternatively, by impairment in
the recovery from ischemia. On the other hand, Fried et al.
(14) found that 48 h after warm ischemia, renal
function and renal histology in uninephrectomized rats were similar to
those observed in the left kidney of rats subjected to bilateral warm
renal ischemia by aortic clamping. Interestingly, in this study, both
groups had in common that all renal tissue was made ischemic.
Considering this background, we started our experiments by testing
whether acute renal damage induced by warm ischemia was different
in ischemic-uninephrectomized (1WIK group) and
ischemic-nonnephrectomized rats (2WIK group). Our results were in
agreement with the findings by Fried et al. (14) and
showed that at 24 h, but also at 7 days, after warm ischemia the
severity of acute tubulointerstitial damage was similar in the 1WIK and
2WIK rats. Furthermore, our findings extend this previous work by
illustrating that the degree of apoptosis induced by warm ischemia was
also similar in 1WIK and 2WIK rats. This fact suggests that in the
early phase after reperfusion, at least when all renal tissue is made
ischemic, nephron mass does not influence the severity of tubular
apoptosis. Considering all these pathological similarities, it seems
that the higher 7-day creatinine clearance in 2WIK rats can therefore
be just a reflection of the presence of two equally injured kidneys,
providing twice the clearance of a single similarly damaged kidney.
Interestingly, despite this similar early pathological outcome, the
long-term consequences of the ischemic insult were quite different in
uninephrectomized and nonnephrectomized rats.
After recovery of animals from postischemic acute renal failure,
creatinine clearance in 1WIK rats became similar to that in the 2WIK
group. Taking into account that these rats with two kidneys obviously
had double the number of nephrons as those uninephrectomized, we can
assume that single-nephron GFR was higher in the 1WIK than in the 2WIK
animals, as previously described (1). Azuma et al.
(2) proposed that ischemia-reperfusion injury causes, in uninephrectomized rats, further reduction of nephrons, enough to
accentuate the glomerular injury by glomerular hyperfiltration and
hypertension, which finally promote the development of
glomerulosclerosis (4, 5). Thus, following
these hyperfiltration caveats, it appears that there is some limit
below which a reduced nephron number lead to progressive renal failure.
Indeed, in our study neither uninephrectomy (the 1NK group) nor warm
ischemia (the 2WIK group) alone was a serious enough injury to exceed
this limit and promote renal scarring. In other words, to induce severe
long-term chronic renal damage both insults are required at one time.
In parallel with this situation of hyperfiltration, 1WIK rats developed significant and progressive proteinuria, whereas in 2WIK rats the
evolution of proteinuria was similar to that shown by 1NK and 2NK rats.
The concept that increased glomerular filtration of protein per se
causes renal scarring in proteinuric nephropathies has been recently
reinforced (27). It has been proposed that increased
filtration of plasma proteins across the glomerular barrier produces
tubular cell damage (28, 38). These injured tubular cells release cytokines and growth factors that promote interstitial inflammation, proliferation of fibroblasts, extracellular matrix accumulation and, finally, renal fibrosis (38). An
elegant explanation that connects the hyperfiltration and proteinuric theories has been suggested by Gandhi et al. (15) by using
a model of renal ablation. These authors proposed that as tubules are
lost (such as a result of ischemia-reperfusion injury), the glomeruli
that retain their tubule connections hypertrophy and filter more
proteins, which accelerate and self-perpetuate the loss of renal
function after renal ablation.
Two main reasons made us consider TGF-
1 as a potential
pathological mediator in this experimental setting. First, it is well documented that this cytokine plays a role in the process of tissue repair after renal warm ischemia (3) and, second, its
sustained expression exerts an important role in the development of
progressive glomerulosclerosis (19) and interstitial
fibrosis (12). Indeed, studies in mesangial cells have
revealed that mechanical stress, as it occurs under hyperfiltration,
leads to matrix accumulation by induction of TGF-
1
(36). Also, nephron mass reduction increases the
generation of angiotensin II, which enhances TGF-
1 gene
expression in tubular epithelial cells (35). Finally,
macrophages that infiltrate the renal interstitium after renal mass
ablation synthesize TGF-
1 (12), which,
acting on fibroblasts, induces interstitial fibrosis. In our study,
1WIK rats disclosed TGF-
1 overexpression before the appearance
of chronic renal failure and severe renal lesions. Moreover, only
this group of animals showed a sustained and progressive renal
TGF-
1 upregulation. On the contrary,
TGF-
1 mRNA did not increase in the 2WIK group and,
moreover, its expression was similar to that in 2NK rats. We can
speculate that after warm ischemia the initial increase in
TGF-
1 involved in restoration of tubular integrity is
further enhanced by uninephrectomy, either by means of enhancing
angiotensin II or by glomerular hypertension. Therefore, the presence
of two kidneys, even though they have suffered from a severe
ischemia-reperfusion injury, is not associated with
TGF-
1 overexpression.
In summary, this study shows for the first time that, in the absence of
renal ablation, bilateral warm renal ischemia does not induce long-term
TGF-
1 upregulation, severe chronic renal lesions, and
chronic renal failure. Our results provide a rationale for
investigating, in an allogeneic transplant setting, whether double-kidney allografting would be a potential way of improving the
poor long-term results of non-heart-beating renal transplantation in
the case of prolonged warm renal ischemia.
 |
ACKNOWLEDGEMENTS |
We are deeply grateful to Margarita Carmona for performing
histological techniques.
 |
FOOTNOTES |
This work was supported by Fondo de Investigaciones Sanitarias (FIS)
Grants 98/0756 and 98/0029-02 and Uriach Laboratories, Barcelona,
Spain. J. M. Cruzado is a fellow from CIRIT Comissio Interdepartamental per a la Recerca i Innovació
Tecnológica. (FIAP 96/9207). I. Herrero and M. Riera are
fellows from Fundaci-Catalana de Transplantament. N. Lloberas is
supported by a grant from Fundaci-August Pi i Sunyer, L'Hospitalet, Spain.
Address for reprint requests and other correspondence: J. M. Grinyó, Nephrology Dept., Hospital de Bellvitge, Feixa Llarga s/n, 08907 L'Hospitalet, Barcelona, Spain (E-mail:
jmgrinyo{at}medicina.ub.es).
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.
Received 18 October 1999; accepted in final form 22 March 2000.
 |
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