Trimetazidine Reduces Renal Dysfunction by Limiting the Cold Ischemia/Reperfusion Injury in Autotransplanted Pig Kidneys
THIERRY HAUET*,,
JEAN-MICHEL GOUJON*,
ALAIN VANDEWALLE,
HERVE BAUMERT*,
LOUIS LACOSTE*,
JEAN-PAUL TILLEMENT§,
MICHEL EUGENE and
MICHEL CARRETIER*
*Unité de Chirurgie
Expérimentale,
Département de
génétique
animale, Institut National de la Recherche Agronomique (INRA), Le Magneraud,
Surgères and GRTMV,
Faculté de
Médecine, Centre Hospitalo Universitaire, La
Milétrie, Poitiers, France Laboratoire de RMN et Explorations Fonctionnelles,
Hôpital Saint-Louis, Paris, France Institut National de la Santé et de la
Recherche Médicale,
Unité U478, Institut
Fédératif de
Recherche 02, Faculté de
Médecine Xavier Bichat, Paris,
France §Laboratoire de Pharmacologie, Faculté de
Médecine, Paris XII,
Créteil, France.
Correspondence to Dr. Thierry Hauet, Unité de
Chirurgie Expérimentale,
Département de
génétique
animale, Institut National de la Recherche Agronomique, Le Magneraud, B.P. 52,
17000 Surgères, France. Phone: +33 5 46 68 30
56; Fax: +33 5 46 68 30 87; E-mail:
hauet{at}magneraud.inra.fr
Abstract. Ischemia/reperfusion injury leads to delayed graft
function,which is a major problem in kidney transplantation. This study
investigatedthe effects of adding trimetazidine (TMZ) to the perfusate of
cold-storedkidneys on the function of reperfused autotransplanted pig kidney.
Theleft kidney was removed and cold-flushed with Euro-Collins (EC),or
University of Wisconsin (UW) solutions with or without 10-6MTMZ
and stored for 48 h at 4°C. The kidneys were then autotransplantedand the
contralateral kidneys were removed. Several parameterswere analyzed over the
14 d after transplantation. The survivalrate was 57% in pigs transplanted
with kidneys cold-flushedwith UW and 43% for those flushed with EC solution;
it was 100%for pigs having kidneys cold-flushed with TMZ-supplemented UWand
EC solutions. The functions of the transplanted kidneyswere also better
preserved after cold flush with TMZ-supplementedsolutions than with TMZ-free
solutions. Creatinine clearancewas higher and the urinary excretion of
trimethylamine-N-oxideand dimethylamine, used as markers of renal
medulla injury,were lower in animals transplanted with kidneys cold-flushed
withTMZ-supplemented solutions than with TMZ-free solutions. The
cytoprotectiveaction of TMZ also reduced interstitial and peritubular
inflammationand the numbers of infiltrating mononuclear CD45+ and
CD3+ Tcells. These results indicate that the tissue damage due to
ischemia/reperfusioninjury may be prevented, at least in part, by adding TMZ
topreservation solutions.
The renal damage resulting from ischemia and reperfusion mayhave adverse
effects on the outcome of organ transplants, increasingboth posttransplant
morbidity and the early loss of transplantedkidneys. Ischemia/reperfusion
injury is not uncommon in cadavericrenal transplantation; it adversely
affects early graft functionand influences the development of chronic graft
dysfunction
(1,2).
Althoughthe impact of delayed graft function (DGF) on graft survivalis
controversial
(3,4),
several studies have shown that DGFand acute rejection episodes may have
additive adverse effects
(5,6,7).
Improvementsin cold preservation techniques and solutions, such as the
Universityof Wisconsin (UW) solution, have greatly facilitated the useof
organs, including kidneys, for transplantation. However,the time of cold
ischemia still has an important influence onDGF
(8). Cold storage and
reperfusion both cause loss of cellpolarity, disruption of the cytoskeleton,
and perturbationsin the polarized membrane transport proteins in postischemic
kidneys
(9,10).
Reperfusion injury is the result of a cascade of events causingthe
allograft to become inflamed. There is evidence indicatingthat the
reperfusion of ischemic tissue leads to a rapid, nonspecificinjury of
allogenic renal grafts in which adhesion moleculesand neutrophil adhesion are
upregulated
(1,2).
Mononuclear cells(T cells and monocyte/macrophages) infiltrate postischemic
ratkidneys, and the expression of T cell-associated cytokines,
monocyte/macrophageactivation products, and MHC class II antigen are
upregulatedafter cold ischemia/reperfusion injury
(11,12).
However, theearly and late consequences of ischemia/reperfusion injury canbe
prevented by soluble P- and E-selectin ligand
(11), or byblocking T cell
costimulatory activation (12)
in the absenceof alloantigen. Injury acts as an adjuvant that increases the
expressionof MHC antigens on epithelial and endothelial cells, and stimulates
therecruitment and activation of antigen-presenting cells
(13).Thus, the synergy
between initial delayed function and acuterejection of renal allograft
appears to be due to the increasedimmunogenicity of the grafted organ, which
promotes a nonspecifichost inflammatory response and amplifies the
cytokine-adhesionmolecule cascade of immune injury.
The anti-ischemic drug trimetazidine (TMZ) has been shown toimprove the
function of isolated perfused pig kidneys exposedto prolonged cold ischemia
(14). This drug impairs lipid
peroxidationand reduces intracellular acidosis during the cold storage and
reperfusionof isolated rat kidneys
(15). TMZ also protects the
functionsof mitochondria from rat liver subjected to ischemia (30 min)and
reperfusion (16). High- and
low-affinity [3H]-TMZ bindingsites have recently been found on rat
liver outer and the innermitochondrial membrane leaflets
(17). The deleterious effects
ofischemia/reperfusion are correlated with altered mitochondrialfunction,
which can be prevented by TMZ
(14,15,16).
This studywas carried out to determine whether adding the anti-ischemicTMZ
drug to preservation solutions limits renal cold ischemiainjury and
reperfusion damage in autotransplanted pig kidneys.
Animals
All experiments were conducted on large white male pigs weighing42 to 51
kg (Institut National de la Recherche Agronomique,Le Magneraud,
Surgères, France). The pigs were housed
accordingto University and National guidelines and kept in a conventional,
closedhousing system at 22 ± 2°C, with a relative humidityof 75
± 5% and 24-h low intensity light. They were transferredto a special
housing unit 24 h before surgery where they werekept singly in pens. They
were fed a piglet diet (INRA 2213,Arrive, Saint Fulgent, France) and had free
access to tap water.
Surgical Procedures
All surgical procedures were performed under sterile conditionsduring
normal daylight hours. The experimental design was aprospective randomized
double-blind trial with strict exclusioncriteria: Animals that died from
causes other than renal failureduring the 2-wk follow-up period and animals
that developedrenal artery, renal vein, or ureteric occlusions were excluded.
Foodwas withheld overnight before surgery. Each pig was tranquilizedby a
rapid, nontraumatic nasal administration of 0.2 mg/kg midazolam(Laboratoire
Roche, Neuilly-sur-Seine, France) and underwentgeneral anesthesia by halothan
(Laboratoire Belamont, Paris,France) and 100% oxygen 20 min later. A 20-gauge
plastic catheter(Becton Dickinson Vascular Access Inc., Sandy, UT) was
insertedinto an ear vein. Atropine sulfate (10 µg/kg) was given
intravenouslyto reduce pharyngeal and tracheal secretion and prevent
postintubationbradycardia. The left kidney was reached through a midline
abdominalincision. The left renal vascular pedicle and the ureter were
atraumaticallyisolated, and 100 U/kg heparin was infused intravenously 10min
before nephrectomy. The removed kidney was immediately flushedwith the iced
preservation solutions described below and storedat 4°C for 48 h. It was
then returned to the animal by heterotopicautotransplantation via the midline
incision. End-to-side aortaand inferior vena cava anastomoses, just
above the iliac bifurcation,were performed using 5.0 polypropylene sutures
(Prolene, Ethicon,Neuilly, France). Ureteroneocystostomy was performed and
thecontralateral kidney was removed.
Experimental Protocols
The removed kidneys were immediately flushed with 300 to 500ml of iced
Euro-Collins (EC) or UW solutions with or without10-6 M TMZ
(provided by the Institut de Recherche InternationalServier, Paris, France).
TMZ was added to the preservation solutionsjust before flushing. The
concentration of TMZ used (10-6 M)was similar to the Ka of the
high-affinity TMZ binding sitesin rat liver mitochondria
(17). Previous studies have
shownthat this drug concentration is beneficial in various isolatedperfused
heart and kidney systems
(12,13,18,19,20).
The pigswere divided into five groups as follows: control group,
uninephrectomizedpigs (n = 4); group A, transplanted kidneys
cold-flushed withthe EC solution alone (n = 8); group B,
transplanted kidneyscold-flushed with the TMZ-supplemented EC solution
(n = 8);group C, transplanted kidneys cold-flushed with the UW
solutionalone (n = 7); and group D, transplanted kidneys
cold-flushedwith the TMZ-supplemented UW solution (n = 7). Plasma
and urinarycreatinine (Cr) were measured enzymatically (Crea, Johnson and
Johnson,Rochester, NY) to determine the creatinine clearance (CCr)
2d before surgery and 1, 3, 5, 7, 11, and 14 d after transplantation.
Proton Nuclear Magnetic Resonance Spectroscopy
The amounts of trimethylamine-N-oxide (TMAO) and dimethylamine
(DMA)in the urine and TMAO in the plasma (TMAOp) were analyzed usingproton
nuclear magnetic resonance (NMR) spectroscopy. Bloodwas collected on
Na+ fluoride-oxalate and centrifuged at 4000rpm for 10 min, and
the plasma was stored frozen at -20°C.Aliquots of urine (0.45 ml)
collected for 24 h were frozen at-20°C. The urine samples were thawed at
room temperature,placed in 5-mm NMR tubes, and mixed with 0.05 ml of
deuteriumoxide solution containing sodium d-(trimethylsilyl)
propionate.Spectra were acquired at 400 MHz on a spectrometer equippedwith a
sample changer (Brucker AM 400WB, Paris, France). Thechemical shifts were
referenced to the internal sodium d-(trimethylsilyl)propionate
resonance at 0 parts per million (ppm). The watersignal was suppressed using
the presaturation technique for2 s at 0.08 W. The sweep width for plasma
samples was 6000 Hz,the pulse was 60°C, and eight scans were accumulated.
Thesweep width for urine samples was 6000 Hz, the pulse was 60°C,and 32
scans were accumulated. The NMR signals were Fourier-transformedwithout any
window function. The aliphatic region of the spectra(0.5 to 4.4 ppm) was
plotted in absolute intensity mode. Resonanceswere identified from the recent
literature chemical-shift data,or by adding standard compounds. The ratios of
TMAO and DMAwere calculated from the urine NMR spectra and expressed as
µM/Cr,corresponding to the ratio of µM products over plasmaCr (in M).
The occurrence of the TMAO resonance (TMAOp) in plasmaspectra was noted only
when it was intense enough to be separatedfrom glucose resonances.
Histologic Studies
Kidneys were processed for light and electron microscopy. Biopsytissue
samples from the deep cortex-outer medulla region ofthe kidney were fixed in
Dubosq-Brazil and 10% formalin in phosphate-bufferedsaline (PBS), embedded in
paraffin, and stained with hematoxylinand eosin, periodic acid-Schiff. Tissue
sections (5 µm)were examined by two pathologists blinded to the
experimentalconditions. Small pieces of renal tissue were fixed in 2.5%
glutaraldehyde,washed and post-fixed in 2% osmium tetroxide for 2 h at
4°C,dehydrated in a graded series of ethanols, and embedded in araldite
fortransmission electron microscopy. Ultrathin sections were cutand stained
with uranyl acetate and lead citrate, and examinedunder an electron
microscope (JEOL 100 CX, Tokyo, Japan). Thedegree of histologic lesioning was
determined using a semiquantitativegraded scale: 0, no abnormality; 1, mild
lesions affecting <25%of kidney samples; 2, lesions affecting 25 to 50% of
kidneysamples; 3, lesions affecting 50 to 75% of kidney samples; 4,lesions
affecting >75% of kidney samples. These scores wereadapted from the BANFF
classification
(21,22).
Immunohistochemical Studies
The cells infiltrating the interstitium and the peritubularareas were
phenotyped using indirect immunohistochemistry. Kidneysections (5 µm) from
biopsies taken 3 and 14 d after autotransplantationwere incubated with mouse
monoclonal antibodies (dilution 1:20)against the porcine leukocyte common
antigen CD45 (Serotec ProductData Sheet, Oxford, United Kingdom), the porcine
MC1218 macrophage/monocyteand neutrophils marker (Serotec Product Data
Sheet), or thehuman CD20 B cell marker, which cross-reacts with porcine B
cells(Dako, Copenhagen, Denmark), and with the rabbit polyclonalantibody
against the human CD3 pan-T cell marker (1:40), whichcross-reacts with
porcine T cells (Dako) for 30 min at roomtemperature. As controls, indirect
immunocytochemistry usingthe MC1218 antibody was performed on swine blood
mononuclearcells obtained by Ficoll-Hypaque (Pharmacia, Piscataway, NJ)
densitygradient centrifugation
(23) and by testing the
anti-CD20 andMC1218 antibodies on sections of pig abdominal lymph nodes.In
all cases, the sections were rinsed in PBS and incubatedwith biotinylated
anti-species (mouse or rabbit; Dako) IgG (1:100)for 20 min at room
temperature, rinsed again in PBS, and incubatedwith alkaline
phosphatase-conjugated streptavidin (Dako) for20 min (1:100) at room
temperature. Phosphatase alkaline activitywas demonstrated by staining with a
freshly prepared substratesolution of Fast Red (Sigma, Saint Louis, MO) in
Tris-bufferedsaline. Sections were counterstained in hematoxylin and mounted
inAquamount. All sections were examined under blinded conditionsand
photographed.
Statistical Analyses
Values are given as means ± SEM. Statistical differencesbetween
groups were calculated by unpaired t test or ANOVA.The
Student-Neuman Keuls test was used for multiple comparisonanalyses, and the
Kruskall-Wallis test was used for nonparametricanalyses. A P value
<0.05 was considered significant.
Effect of TMZ on Survival and Creatinine Clearance
A total of 32 pigs underwent kidney transplantation. Two animalswere
excluded from the study, one from the group A (TMZ-freeEC perfusion
condition) because of the occurrence of gastriculcerations and the other from
the group B (TMZ-supplementedEC perfusion condition) because of ureteric
obstruction. Eachexperimental group was well standardized in terms of total
bodyand kidney weights (Table
1). The outcomes after autotransplantationand contralateral
nephrectomy differed markedly. Survival was100% in the control group, 43% in
group A, and 57% in groupC; the two groups with autotransplanted kidneys
cold-flushedwith TMZ-free EC or UW solutions. Pigs died on postoperativedays
3 and 5 in group A and on postoperative days 6 and 11 ingroup C. All of these
animals developed acute renal failure,confirmed by histologic analysis (data
not shown). All of thepigs autotransplanted with kidneys cold-flushed with
TMZ-supplementedEC and UW solutions (groups B and D) were alive (100% survey)
14d after surgery.
Table 1. Pig body weights and autotransplanted kidney
weightsa
The CCr was identical in all experimental groups before surgery
(Figure 1).The levels of
CCr from the uninephrectomized animals (controlgroup) were lower
than in intact animals but remained stable(100 to 125 ml/min) over the 2 wk
after surgery (Figure 1, top).
TheCCr in all autotransplanted groups was much lower
(Figure 1,bottom). However,
the CCr levels for the pigs autotransplantedwith kidneys
cold-flushed with the TMZ-supplemented EC or theTMZ-supplemented UW solution
were significantly higher thanthose for the pigs autotransplanted with
kidneys cold-flushedwith TMZ-free EC or TMZ-free UW solutions
(Figure 1, bottom).The
CCr in these experimental groups gradually increased fromday 5 to
day 14 after surgery, and the highest CCr occurredin experimental
group D (kidneys cold-flushed with the TMZ-supplementedUW solution). Although
mild proteinuria was detected in allgroups of transplanted animals 14 d after
surgery, it was alsolower in the groups of pigs autotransplanted with kidneys
cold-flushedwith the TMZ-supplemented solutions (group B, 0.8 ± 0.4;
groupD, 0.9 ± 0.3 g/24 h) than with the TMZ-free solutions(group A,
2.1 ± 0; group C, 1.5 ± 0.1 g/24 h).These first results thus
validated the surgical protocol used,since the CCr from
uninephrectomized animals (control group)remained stable over the entire
course of the study, and alsoindicated that the addition of TMZ to the
preservation solutionameliorates the CCr from transplanted
kidneys.
Figure 1. GFR of unilateral nephrectomized pigs and pigs with autotransplanted
kidneys. Creatinine clearance was measured in animals 2 d before surgery (-2)
and 1 to 14 d after uninephrectomy (top panel, control group) or
autotransplantation (bottom panel). Autotransplanted kidneys were cold-flushed
with Euro-Collins solution (EC), EC plus trimetazidine (TMZ) solution,
University of Wisconsin solution (UW), or UW plus TMZ solution.
*P < 0.05; **P < 0.01.
Effect of TMZ on Renal Excretion of TMAO and DMA
The delayed graft function caused by ischemia/reperfusion injuryis often
linked to renal medullary cell damage
(24,25,26).
Theratios of urinary TMAO and DMA excretion over plasma creatininewere
analyzed by NMR spectroscopy to determine the influenceof
ischemia/reperfusion on renal medulla injury. The plasmaTMAO (TMAOp) was
undetectable in intact pigs and barely detectablein uninephrectomized animals
(control group); it increased greatlyin pigs autotransplanted with kidneys
cold-flushed with TMZ-freeEC and TMZ-free UW solutions (groups A and C)
during the 2 wkafter surgery (Figure
2). The TMAOp of pigs autotransplantedwith kidney cold-flushed
with the TMZ-EC (group B) or TMZ-UW(group D) solutions was significantly
lower than in pigs withkidney cold-flushed with TMZ-free EC or TMZ-free UW
solutions(Figure 2). The group
D pigs (kidneys cold-flushed with TMZ-UW)had lower TMAOp than the pigs in
groups A, B, and C (Figure 2).Consistent with these results, the urinary excretion ofTMAO, expressed as
TMAO (mM):Cr (M) ratio, and the urinary excretionof DMA, expressed as DMA
(mM):Cr (M) ratio, were significantlygreater in pigs transplanted with
kidneys cold-flushed withTMZ-free EC or UW solutions than in those
transplanted withkidneys cold-flushed with TMZ-EC or TMZ-UW solutions
(Figure 3, A and B).These
results suggest that TMZ helps to protectkidney cells during cold
preservation and reperfusion.
Figure 2. Trimethylamine-N-oxide (TMAO) in the plasma of autotransplanted
pigs. Plasma TMAO (TMAOp) was measured by nuclear magnetic resonance (NMR)
spectroscopy in pigs autotransplanted with kidneys initially cold-flushed with
EC, EC plus TMZ, UW, or UW plus TMZ solutions 1 to 14 d after surgery. TMAOp
was not detected (ND) in any of the pigs 2 d before surgery (-2).
***P < 0.001.
Figure 3. Urinary excretion of TMAO and dimethylamine (DMA) in autotransplanted pigs.
Urinary excretion of TMAO (A) and DMA (B) was measured in unilaterally
nephrectomized pigs (Control) and in pigs with autotransplanted kidneys
initially cold-flushed and preserved in EC, EC plus TMZ, UW, or UW plus TMZ
solutions, 1 to 14 d after surgery. *P < 0.05;
**P < 0.01; ***P < 0.001.
Effects of TMZ on the Morphology of Reperfused Kidneys
Biopsies from the deep cortex-outer medullary regions were taken40 min
after reperfusion. They showed considerable cell damageand cell debris in the
tubular lumens of the kidneys initiallycold-flushed with the EC and UW
preservation solutions (Figure 4, A and
B).The tissue architecture of all reperfused kidneysinitially
cold-flushed with TMZ-free UW solution
(Figure 4B)looked better than
those flushed with TMZ-free EC solution
(Figure 4A).The tubule
morphology of kidneys flushed with TMZ-EC orTMZ-UW was much better conserved
(Figure 4, C and D). Again,the
reperfused kidneys cold-flushed with TMZ-UW had better morphologicshapes
(Figure 4D) than those
preserved with TMZ-EC (Figure
4C).The electron microscopy pictures also showed a marked
reductionin the density of apical brush borders of proximal tubule cells(S3
segments from deep cortex-outer medulla region) in reperfusedkidneys
initially cold-flushed with TMZ-free EC and UW solutions
(Figure 5, A and B).In
contrast, the reperfused kidneys from groupsB and D, cold-flushed with TMZ-EC
(Figure 5C) or TMZ-UW
(Figure 5D),had well-organized
proximal apical brush borders. Theseresults agree with observations showing
that TMZ can protectcold-preserved renal proximal tubule cells
(14,15).
Figure 4. Influence of TMZ on kidney morphology. Light microscopy photographs of deep
cortex-outer medullary kidney biopsy samples taken 40 min after the
autotransplantation of kidneys cold-flushed with EC (A), UW (B),
TMZ-supplemented EC (C), or TMZ-supplemented UW (D) solutions and stored for
48 h at 4°C before surgery. There are numerous tubule cell lesions and
debris in the lumens of tubule sections (tl) of kidneys initially cold-flushed
with TMZ-free EC (A) and TMZ-free UW (B) solutions. The tubule sections
appeared to be better preserved in kidneys cold-flushed with TMZ-supplemented
EC (C) or TMZ-supplemented UW (D) solution. Magnification, x312.
Figure 5. Influence of TMZ on the integrity of proximal tubule brush borders.
Electron micrographs of proximal tubule sections from biopsies taken 40 min
after the autotransplantation of pig kidneys initially cold-flushed with EC
(A), UW (B), TMZ-supplemented EC (C), or TMZ-supplemented UW (D) solution and
stored for 48 h at 4°C before surgery. Proximal tubule cells from kidneys
cold-flushed and preserved with the TMZ-supplemented EC (C) and UW (D)
solutions bore more densely packed apical microvilli than the proximal tubule
cells from kidneys cold-flushed and preserved with TMZ-free EC (A) or TMZ-free
UW (B) solutions. B, brush border; tl, tubular lumen. Magnification,
x3300.
Table 2 summarizes the
degrees of cellular alteration observedin the autotransplanted kidneys just
after their reimplantation(40 min after reperfusion) and 14 d after
transplantation. Therewere marked tubular lesions in the reperfused kidneys
from groupsA and C (TMZ-free EC or TMZ-free UW). The cell changes in
reperfusedkidneys cold-flushed with TMZ-supplemented solutions were
significantlyless marked (Table
2). The semiquantitative scores obtained14 d after
autotransplantation also showed that there was lesscell damage in the pigs
autotransplanted with kidneys initiallycold-flushed with TMZ-UW or TMZ-EC
(Table 2). The scores for
kidneyspreserved in TMZ-free or -supplemented UW solutions (groupsC and D)
were also significantly lower than those for TMZ-freeor -supplemented EC
solutions (groups A and B). The rare glomerulilocated in the deep cortex were
not altered in any of the experimentalgroups. Thus, these morphologic data
indicate that TMZ had beneficialeffects on the ischemia-reperfusion caused by
cold storage.
Table 2. Histologic damage scores in autotransplanted pig
kidneysa
Effects of TMZ on Cellular Infiltration in Autotransplanted Pig
Kidneys
We next analyzed the correlation between better conservationof whole
kidney functions and morphology produced by addingTMZ to the preservation
solutions and cellular inflammation.The histology of biopsies taken 14 d
after transplantation showedareas of large polymorphic cell infiltrates with
abundant eosinophiliccytophilic cytoplasm and round, indented, or irregular
nucleiwith clumped chromatin in all experimental groups
(Figure 6).However, the
infiltration was more pronounced in posttransplantedkidneys initially
cold-flushed with EC preservation solutionalone
(Figure 6A) than in those
perfused with UW solution alone(Figure
6B). It was markedly lower in posttransplanted kidneys
cold-flushedand stored in TMZ-supplemented solutions
(Figure 6, C and D).Indirect
immunofluorescence with leukocyte, macrophage/monocyteand neutrophils, and B
and pan-T cell markers was used to identifythe cell types involved. We first
checked that the anti-CD20and MC1218 antibodies cross-reacted with pig
tissues. Swinelymph node sections were positively stained with these two
antibodies(Figure 7, A and B).
Swine blood mononuclear cells were alsostained with the MC1218 antibody
(Figure 7C). There was never
positivestaining using the anti-CD20 B cell antibody on kidney biopsiestaken
3 d (data not shown) and 14 d after transplantation
(Figure 7, D and E).Positive
staining with the MC1218 macrophage/monocyteand monocyte antibody was
detected in all kidney biopsies taken3 d after transplantation. There was
more MC1218-positive cellsin posttransplanted kidneys from groups A and C,
initially cold-flushedwith TMZ-free EC and TMZ-free UW solutions
(Figure 7, F and G),than in
kidneys from groups B and D (data not shown). Incontrast, no more positive
staining was observed in all thekidney biopsies performed 14 d after
transplantation (Figure 7, H and
I).However, all of the 14-d transplanted kidneys containedareas
of cell membrane labeled with the anti-CD45 leukocyteantigen antibody
(Figure 8, A through D).
CD45-positive cellswere more abundant in post-transplanted kidneys from
groupsA and C (Figure 8, A and
B) than in kidneys from groups B andD
(Figure 8, C and D), in
agreement with the semiquantitativescores
(Table 2). Immunostaining with
the anti-CD3 pan-T cellantibody was also intense
(Figure 8, E through H). Here
again,there were more positive cells in the transplanted kidneys fromgroups
A and C (Figure 8, E and F)
than in the kidneys fromgroups B and D
(Figure 8, G and H). Thus,
there were T lymphocytecells in the cellular infiltrates, but no B cells or
monocytes/macrophageswere detected 2 wk after the transplantation.
Figure 6. Cell infiltration in autotransplanted pig kidneys showing the cellular
infiltration 2 wk after transplantation. Kidney sections stained with
hematoxylin and eosin show that the cell infiltrates in the interstitial and
peritubular areas were more marked in transplanted kidneys initially
cold-flushed with TMZ-free EC (A) and UW (B) solutions than in kidneys
initially cold-flushed with TMZ-supplemented EC (C) or UW (D) solutions.
Magnification, x200.
Figure 7. Monocytes and macrophages in autotransplanted pig kidneys. Indirect
immunofluorescence using anti-CD20 B cell (A) and anti-porcine MC1218
macrophage/monocyte and neutrophil (B) antibodies showed positive cell
staining (appearing in red) in swine lymph node sections. Mononuclear cells
from swine blood were also stained with the MC1218 antibody (C).
Immunocytochemistry using the anti-CD20 antibody showed no staining of tissue
sections from the 14-d posttransplanted kidneys initially cold-flushed with
TMZ-free EC (D) or UW (E) solutions. Areas of positive-stained cells with the
MC1218 antibody were detected in the 3-d posttransplanted kidneys initially
cold-flushed with TMZ-free EC (F) or UW (G) solutions. No more MC1218-positive
cells were detected in the 14-d posttransplanted kidneys initially
cold-flushed with TMZ-free EC (H) or UW (I) solutions. Magnification,
x200.
Figure 8. Identification of T cells in autotransplanted pig kidneys. Indirect
immunofluorescence using anti-leukocyte common antigen CD45 (A through D) and
anti-CD3 pan-T cell (E through H) antibodies was done on tissue sections from
14-d posttransplanted kidneys. More CD45-positive cells (appearing in red)
were detected in kidneys initially cold-flushed with the TMZ-free EC (A) or UW
(B) solutions than in those cold-flushed with TMZ-supplemented EC (C) or UW
(D) solutions. Similar differences in the number of CD3-positive cells
(appearing in red) were observed between tissue sections from kidneys
preserved with TMZ-free EC (E) or UW (F) and tissue sections from kidneys
preserved with TMZ-supplemented EC (G) or UW (H) solutions. i, cell
infiltration. Magnification, x125.
The results clearly show that adding TMZ to preservation solutionsgreatly
improves the function of autotransplanted pig kidneys.This anti-ischemic drug
also protects kidney tubule epithelialcells and reduces the cellular
inflammation caused by ischemia/reperfusioninjury.
Ischemia favors the depletion of cellular adenosine nucleotides,
alterationsin membrane ATP-dependent ionic transporters, and the
intracellularaccumulation of Ca2+, Na+, and water. The
great swelling ofendothelial and tubular epithelial cells due to ischemia not
onlyincreases the acidosis caused by anaerobic oxidation, but alsoalters
cell permeability (27) and
favors the obstruction ofcapillary flow. Outer medullary vascular congestion
is a prominentfeature of ischemic acute renal failure and transplanted
kidneysdamaged during cold preservation of the grafts
(27,28).
Theoutflow of blood from the medulla during reperfusion is blocked
(29),limiting oxygenation of
the tubule epithelial cells locatedin this region
(30). The reperfusion of
ischemic tissues alsoincreases the release of intracellular enzymes, the
influx ofintracellular Ca2+. Free radicals and other reactive
oxygenspecies that trigger T cell activation
(1) are produced aftercold
ischemia and rewarming during reperfusion. The increasein intracellular
Ca2+ activates membrane phospholipase A2. Theoxygen
supplied by blood reperfusion generates free oxygen radicals,which react with
lipid cellular membranes. The peroxidationof cell membrane lipids can disrupt
the balance of vasoactiveeicosanoid metabolism, leading to vasoconstriction
due to excessthromboxane synthesis, and a decrease in the production of
prostacyclinand prostaglandin I2
(31). The release of
proteases, inflammatorycytokines, chemoattractants, and growth factors such
as fibrogenicgrowth factor TGF-ß is also associated with upregulationof
adhesion molecules and activation of leukocytes, macrophages,and monocytes in
postischemic reperfused kidneys
(1,32).
We and others have recently reported that TMZ added to the perfusion
solutionsmarkedly improves resistance to hypoxic stress and reduces cold
ischemia/reperfusioninjury in ex vivo and in vitro organs
(14,15,33,34).
These studiesprovide evidence that pharmacologic concentrations of TMZ in
preservationor perfusion solutions maintain Ca2+ homeostasis and
preservemitochondrial function during prolonged cold storage. TMZ also
protectsagainst cold damage due to ischemia/reperfusion injury in isolated
arrestedrat heart (20) and
isolated ischemic and reperfused rat kidney
(35).Two classes of TMZ
binding sites, thought to be involved inthe mitochondrial permeability
transition pore, have been foundon the inner and outer membranes of purified
rat liver mitochondria(17).
TMZ also counteracts the change in mitochondrial permeabilitycaused by
Ca2+ overload due to the pro-oxidant tert-butylhydroxiperoxide
(36).These results led us to
examine the effects of TMZ on renalfunction in autotransplanted pig kidney.
TMZ seems to improvethe cold preservation of kidneys to be transplanted,
givingbetter survival, recovery of kidney function, and protectionagainst
cold ischemia/reperfusion. The NMR spectroscopy dataindicate that cold
preservation of the kidneys with TMZ-supplementedsolutions markedly reduces
the excretion of the renal medullaryosmolytes TMAO and DMA, used as indexes
of renal medullary damage
(37,38,39,40).
Themorphologic studies also indicate that TMZ protects the proximaltubule
epithelial cells from damage caused by cold ischemiaand reperfusion.
The influx of inflammatory cells, particularly T cells, doesnot appear to
be directly related to organ rejection in thisexperimental model of
"pure" ischemia/reperfusion, but it pointsout the importance of
an influx of inflammatory cells duringischemia/reperfusion injury. Several
processes involved in theactivation of T cells, independently of any
alloimmune stimulus,may influence the cell damage caused by ischemia/injury
(5,32).
Thepresent study extends these observations by showing that prolongedcold
ischemia and autotransplantation lead to the infiltrationof many T cells into
damaged kidney areas. But the infiltrationof T cells into the
autotransplanted pig kidneys is markedlyreduced when TMZ is added to the
preservation solutions. Infiltrationof macrophages and monocytes occurs
shortly (day 3) after theautotransplantation of the pig kidneys, similar to
that reportedin ischemic-reperfused rat kidneys by Takada et al.
(11). However,we failed to
detect any positive-labeled cells with the porcineMC1218 antibody in kidney
biopsies taken after a more prolongedperiod (14 d) following ischemia. Hence,
cryoprotection by TMZimproves the quality of the transplanted kidneys and
reducesthe inflammation caused by ischemia/reperfusion. Ischemia/reperfusion
injurycould perhaps contribute to the modification of antigens intissue
damaged by reactive oxygen metabolites
(32). The antigensin normal
tissue tend to be ignored and the antigens in injuredtissue are likely to
activate an immune response perhaps inpart due to proinflammatory cytokines
(41).
This study demonstrates that TMZ can reduce injury due to prolongedcold
ischemia/reperfusion and preserve renal functions in autotransplantedpig
kidneys. TMZ may thus be a useful drug, either alone orin combination with
adhesion protein-blocking monoclonal antibodiesand/or other recombinant
proteins, against ischemia/reperfusioninjury.
Acknowledgments
This work was supported by grants from the Etablissement
Françaisdes Greffes, the
Ministère de la Recherche (92C0746),the
Ministère de l'Education Nationale (EA 427),
theAssociation pour le Développement de la
Dialyse àDomicile (ADA 17), and the Institut
de Recherches InternationalesServier. We thank W. Hebrard and C. Henry for
technical assistanceand Dr. O. Parkes for editing the English text.
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Received for publication January 28, 1999.
Accepted for publication June 29, 1999.
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Abstract
Introduction
