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American Journal of Transplantation 2009; 9: 114–123
Wiley Periodicals Inc.
C
2009 The Authors
Journal compilation C
2009 The American Society of
Transplantation and the American Society of Transplant Surgeons
doi: 10.1111/j.1600-6143.2008.02500.x
Effects of Donor Age and Cell Senescence on Kidney
Allograft Survival
A. Melka
,
∗,B.M.W.Schmidt
b,H.Braun
c,
A. Vongwiwatanad,J.Urmson
d, L.-F. Zhue,
D. RaynerfandP.F.Halloran
d
a
Division of Pediatric Nephrology, Gastroenterology and
Metabolic Diseases, Children’s Hospital, Hannover
Medical School, Hannover, Germany
b
Department of Nephrology, Hannover Medical School,
Hannover, Germany
c
Division of Pediatric Nephrology, University of
Heidelberg, Heidelberg, Germany
d
Division of Nephrology and Transplantation Immunology,
Department of Medicine, University of Alberta,
Edmonton, Canada
e
Department of Surgery, University of Alberta, Edmonton,
Canada
f
Department of Laboratory Medicine and Pathology,
University of Alberta, Edmonton, Canada
∗
Corresponding author: Anette Melk,
melk.anette@mh-hannover.de
The biological processes responsible for somatic cell
senescence contribute to organ aging and progression
of chronic diseases, and this may contribute to kid-
ney transplant outcomes. We examined the effect of
pre-existing donor aging on the performance of kid-
ney transplants, comparing mouse kidney isografts
and allografts from old versus young donors. Before
transplantation, old kidneys were histologically nor-
mal, but displayed an increased expression of senes-
cence marker p16
INK4a
. Old allografts at day 7 showed a
more rapid emergence of epithelial changes and a fur-
ther increase in the expression of p16
INK4a
. Similar but
much milder changes occurred in old isografts. These
changes were absent in young allografts at day 7,
but emerged by day 21. The expression of p16
INK4a
re-
mained low in young kidney allografts at day 7, but
increased with severe rejection at day 21. Isografts
from young donors showed no epithelial changes and
no increase in p16
INK4a
. The measurements of the al-
loimmune response—infiltrate, cytology, expression
of perforin, granzyme B, IFN-cand MHC—were not
increased in old allografts. Thus, old donor kidneys
display abnormal parenchymal susceptibility to trans-
plant stresses and enhanced induction of senescence
marker p16
INK4a
, but were not more immunogenic.
These data are compatible with a key role of somatic
cell senescence mechanisms in kidney transplant out-
comes by contributing to donor aging, being acceler-
ated by transplant stresses, and imposing limits on the
capacity of the tissue to proliferate.
Key words: Donor age, kidney aging, p16
INK4a
expres-
sion, senescence, transplantation
Received 17 September 2007, revised 18 September
2008 and accepted for publication 09 October 2008
Introduction
The limitation in the supply of organs for transplantation
compels us to transplant organs from older donors, mak-
ing the biology of aging a key question in organ transplan-
tation. Old donor age impairs early and late graft function
and reduces survival of deceased donor kidney transplants
(1–5). In addition, older donor kidneys are often affected
by age-related diseases such as hypertension, diabetes
and arterial deterioration. Kidneys from older donors have
an increased incidence of delayed graft function, poorer
glomerular filtration rates (GFR) at all times, lower graft
survival and, in some studies, an increased frequency of
rejection (4,6). Indeed, concerns about old donor age are
a major reason for discarding organs. Kidneys from old
donors get more rejection, as defined by tubulitis in the
Banff system, suggesting that they evoke more vigorous
host alloimmune responses (7).
The effects of donor organ aging could be related to so-
matic cell senescence, the state of cells and tissues that
have lost their capacity for repair and replication. Somatic
cell senescence could be a unifying theme in organ trans-
plantation, reflecting the inherent limitations on survival,
repair and replication in somatic tissues (8,9). The hypoth-
esis is that each tissue has a finite capacity for survival,
repair and replication; capacity that is used up slowly by
age and rapidly by abnormal stresses from injury and dis-
ease. Thus, pre-existing aging reduces repair and survival
capacity, and peri- and posttransplant stresses use up more
of this capacity, leading to an earlier failure (8).
In vitro
,
there are two models of somatic cell senescence: replica-
tive senescence and stress- and aberrant signaling-induced
senescence (STASIS). Replicative senescence is caused
by telomere shortening and dysfunction and occurs only in
human fibroblasts but not in mouse embryonic fibroblasts
(10,11). Telomere shortening can be prevented by telom-
erase transfection (12). STASIS can be observed in both
cells from species and is associated with an increased
expression of cyclin-dependent kinase inhibitor p16
INK4a
(13–17). STASIS is caused by extrinsic stresses, whereas
114
Donor Age, Cell Senescence and Allograft Survival
replicative senescence is an intrinsic consequence of cell
replication (18).
In vivo
, somatic cell senescence occurs in
kidneys with aging and probably contributes to the phe-
notype of organ aging and age-related diseases. Telom-
ere shortening occurs in human but not in rodent kidneys
(19,20), whereas increased p16
INK4a
expression occurs in
both (21). In addition to its significant positive correlation
with the kidney age, p16
INK4a
expression also correlated to
the histopathological changes seen in older kidneys (21).
Recent publications have added further support to the as-
sociation of p16
INK4a
expression with a widespread impair-
ment in tissue regeneration in rodents (22–24). Because
of the existence of STASIS in both, human and rodent
senescence, we have focused on studying p16
INK4a
ex-
pression as a central signaling protein in this senescence
pathway.
The present mouse studies explored how the behavior
of kidney transplants from older and young mice differs
and examined both the parameters of rejection and the
parenchymal response to operative stress and rejection.
We chose to study 18-month-old CBA kidneys rather than
those of older mice to avoid the characteristic focal and
segmental glomerulosclerosis that spontaneously occurs
in mice and rats at later times. We assessed whether aging
alters the parenchymal response to immune and nonim-
mune stress and immunogenicity. To assess the evolution
of pathology, we used a previously described non-life-
supporting vascularized renal transplantation model in
mice across full MHC barriers (25–27). We investigated
how transplant stresses affect the morphology of the
epithelium and the expression of p16
INK4a
mRNA and
protein.
Materials and Methods
Mice
Male CBA mice were obtained from the National Institute on Aging, aged
rodent colonies, at 3 (young) or 18 months (old) of age. C57Bl/6 (B6) mice
were purchased from Jackson Laboratory (Bar Harbor, ME). All animals
were kept in a facility that is barrier-maintained and specific pathogen-free.
The animals were transplanted within 1 week after arrival in our colony.
All experiments were performed according to the University of Alberta and
University of Heidelberg Animal Policy and Welfare Committee’s animal
care protocols.
Renal transplantation
The donor mice (CBA at age 3 or 18 months) were anaesthetized and the
abdomen was opened through a midline incision. The right kidney was ex-
cised, flushed and preserved in cold saline solution for 30 min. The host
mice (B6 for allogeneic and CBA for syngeneic grafts, at a weight of 21–
23 g and age of 2 months) were similarly anaesthetized and the right native
kidney excised. The donor kidney was anastomosed heterotopically to the
inferior aorta, vena cava and bladder on the right side, without removing
the host’s left kidney (non-life-supporting kidney transplantation). The mice
were allowed to recover and were killed by cervical dislocation at day 7
or 21 following anesthesia. None of the transplanted hosts received im-
munosuppressive therapy. All hosts received prophylactic antibiotics with a
cephalosporine (cefazolin sodium, Novopharm, Stouffville, Ontario, Canada)
at a dose of 100 mg/kg i.m. to prevent wound and urinary tract infection.
Mice with technical complications or pyelonephritis were removed from the
study. We studied 15 young donors (n =7atday7andn=8 at day 21)
and 13 old donors (n =6atday7andn=7 at day 21) for allografts, and
8 young donors (n =4atday7andn=4 at day 21) and 7 old donors (n =
4atday7andn=3 at day 21) for isografts.
Histopathology
Tissue sections (2 lm) were stained with hematoxylin and eosin (H&E)
or with periodic acid-Schiff (PAS). Histopathologic changes were evaluated
based on the Banff classification for transplant pathology (28). The acute
lesions were scored from 0 to 3 based on the percentage of parenchymal
involvement, as described earlier. For interstitial infiltrate: 0 =representing
no or trivial (<10%) interstitial inflammation, 1 =representing 10–25%,
2=representing 26–50% and 3 =representing more than 50% of the total
parenchyma inflamed. For glomerulitis: 0 =representing no glomerulitis,
1=representing glomerulitis in up to 25% of glomeruli, 2 =represent-
ing glomerulitis in 26–75% of glomeruli and 3 =representing glomerulitis
in more than 75% of glomeruli. For tubulitis: 0 =no mononuclear cells
in tubules, 1 =representing foci with 1–4 cells per tubular cross-section,
2=representing foci with 5–10 cells per tubular cross-section and 3 =
representing more than 10 cells per tubular cross-section. Tubulitis was
assessed for nonatrophic and atrophic tubules separately. For vasculitis:
0=no arteritis, 1 =mild-to-moderate intimal arteritis in at least one arterial
cross-section, 2 =severe intimal arteritis with at least 25% luminal area lost
in at least one arterial cross-section and 3 =transmural arteritis and/or arte-
rial fibrinoid change and medial smooth muscle necrosis with lymphocyte
infiltrate in vessel. We also recorded the extent of graft necrosis, throm-
bosis, edema and cast formation as the percentage of total parenchyma
involved. The assessment of chronic changes included interstitial fibrosis
and tubular atrophy. These changes were scored based on the percentage
of parenchymal involvement. For interstitial fibrosis: 0 =representing inter-
stitial fibrosis in up to 5%, 1 =representing interstitial fibrosis in 6–25%,
2=representing interstitial fibrosis in 26–50% and 3 =representing inter-
stitial fibrosis in more than 50% of the cross-section. For tubular atrophy:
the percentage of tubules affected was assessed for the cortical area of
the complete cross-section. Vascular fibrous intimal thickening and arterio-
lar hyaline thickening did not occur in any of the mice and were therefore
not scored.
In addition, on PAS-stained slides, the number of nuclei per tubular cross-
section and the tubular diameter for nonatrophic and atrophic tubules were
assessed. No distinction was made between proximal and distal tubules;
medullary rays were avoided. Both assessments were done in 10 random
high-power fields (HPFs; 400×magnification). For the measurement of
the tubular diameter, pictures from randomly selected areas were taken
using a Nikon Eclipse-1000 digitizing microscope (Nikon, Melville, NY). The
resulting TIFF-files (size 25.6 ×20.48 inches) were processed using Adobe
Photoshop 5.0 (Adobe Systems Canada, Ottawa, Ontario, Canada) to reduce
their size (size 12.8 ×10.24 inches), making them suitable for ImageJ
software (free share software, NIH, Bethesda, MD). The tubular diameter
was measured in inches on these processed pictures.
Antibodies
Hybridoma cell lines were obtained from ATCC (Rockville, MD). Cell lines
producing monoclonal antibodies, 34–4-20S (anti H-2D), 11–5.2.1.9 (anti-
I-Ak), 11–4.1 (anti-H-2K) and 20–8-4S (anti-H-2KbDb) were maintained in
tissue culture in our laboratory. All other antibodies were purchased: anti
I-Ab(Serotec, Raleigh, NC), anti-mouse CD45 (Cedarlane, Hornby, ON), anti-
mouse CD3 (Serotec, Raleigh, NC), anti-mouse CD8 (Serotec, Raleigh, NC),
anti-mouse CD4 (BD Pharmingen, Mississauga, ON), anti-p16
INK4a
(Santa
Cruz Biotechnologies, Santa Cruz, CA) and Ki-67 (clone MIB-1, Dako, Mis-
sissauga, Ontario, Canada).
American Journal of Transplantation
2009; 9: 114–123 115
Melk et al.
Immunohistochemistry
Immunohistochemistry was performed either on frozen or on paraffin-
embedded biopsy sections. Fresh frozen sections were fixed in acetone
and then incubated with normal goat serum. Slides were then incubated
with rat anti-mouse CD45, CD3, CD4 and CD8. Control slides were treated
with PBS. Next, the slides were exposed to the affinity-purified goat anti-rat
IgG F(ab’)2fragment (ICN, Costa Mesa, CA). The slides were finally stained
with 3’3 diaminobenzidine tetrahydrochloride (DAB) and hydrogen perox-
ide for the peroxidase reaction and counterstained with hematoxylin. The
analysis was done by counting 10 random HPFs by a blinded observer.
Immunoperoxidase staining for p16
INK4a
and Ki-67 was performed using
2-lm sections of paraffin-embedded tissue. Briefly, the sections were
deparaffinized and hydrated. In case of Ki-67 staining, antigen retrieval
was performed in citrate buffer. The sections were then immersed in 3%
H2O2:methanol to inactivate endogenous peroxidase and blocked with 20%
normal goat serum. The tissue sections were then incubated for 1 h at room
temperature with the primary antibody (mouse monoclonal antibody for ei-
ther p16
INK4a
or Ki-67) or isotype control and rinsed with PBS. Following
30 min of incubation with the Envision monoclonal system (Dako), the sec-
tions were washed again in PBS. The visualization was performed using
the DAB substrate kit (Dako). The slides were counterstained with hema-
toxylin and were mounted. The percentage of positive nuclei was assessed
for tubules (for both p16
INK4a
and Ki-67), glomeruli (only for p16
INK4a
)and
interstitium (only for p16
INK4a
) by counting 10 random HPFs by a blinded
observer.
RNA isolation
Total RNA was extracted from tissue samples according to a modification of
the method described by Chirgwin et al. (29). Tissues were homogenized in
a polytron in 4 M guanidinium isothiocyanate, and the RNA was centrifuged
witha5.7MCsCl
2cushion. RNA was isolated by phenol/chloroform extrac-
tion. Concentrations were determined by absorbance at 260 nm.
Reverse transcription (RT) and real-time polymerase chain
reaction (PCR)
Transcription into cDNA was done using Maloney murine leukemia virus
(MMLV) RT and random primers (Life Technologies, Burlington, Ontario,
Canada). The principle of real-time quantitative PCR has been described
by Heid et al. (30). cDNA was amplified in an ABI PRISM 7700 Sequence
Detector (Applied Biosystems, Foster City, CA). All samples were done in
duplicates and run in two separate experiments. Sequence-specific primers
and probe (Table S1) for IFN-c, perforin, granzyme B, p16
INK4a
and hypox-
anthine phosphoribosyl transferase (HPRT) were designed using Primer
Express Software (Applied Biosystems). A relative quantification of gene
expression was performed using the Relative Standard Curve method, as
described in the User Bulletin #2 (Applied Biosystems). Briefly, the number
of PCR cycles that are needed to reach the fluorescence threshold is called
threshold cycle (Ct). The Ct value for each sample is proportional to the
logarithm of the initial amount of input cDNA. The calibrator used consisted
of cDNA derived from different tissues and age groups from normal and
transplanted mice. Standard curves were prepared by serial dilutions of the
calibrator for both the gene of interest and the housekeeping gene HPRT.
The dilutions were arbitrarily numbered 3, 1.5, 0.75, 0.375 and 0.1875. The
Ct values for all samples were then assigned an arbitrary value based on
the standard curves. The arbitrary values for the gene of interest and HPRT
were divided (gene/HPRT) in order to normalize to HPRT. Then, the mean
value for the duplicates is calculated. All values are given as a gene of
interest to HPRT ratio.
Statistical analysis
Data analyses were performed using SPSS (SPSS Inc., Chicago, IL). For
quantitative variables, the means among different groups were compared
using ANOVA, and
t
-tests with Bonferroni correction were used for multiple
pairwise comparisons. For ordinal observations, the different groups were
compared using the Kruskal–Wallis test, and pairwise comparisons were
performed using the Mann–Whitney
U
statistics. All values are given as
mean (M) ±standard deviation (SD).
Results
We studied how pre-existing aging may alter the evolu-
tion of rejection on the renal parenchyma and the effect of
stress of the transplantation procedure. The old kidneys
were evaluated in our previously described renal trans-
plant model (25–27). We used a donor–recipient combina-
tion either fully mismatched for MHC and non-MHC anti-
gens (CBA into B6, allogeneic) or fully matched for MHC
and non-MHC antigens (CBA into CBA, syngeneic), with
no immunosuppression. The left host kidney remained in
place to maintain the survival (non-life-supporting trans-
plant model) and health of the host, with no incidental loss
of subjects due to death from uremia and hyperkalemia.
This provided us with the ability to assess the evolution of
pathologic lesions over time.
Histopathology
Normal CBA mice:
Young CBA mice lacked features of
aging or renal disease (Figure 1A and B). Four of the 18
old CBA showed mild patchy tubular atrophy, affecting less
Figure 1: Tubular cross sections in (A) normal young CBA
mice, (B) Normal old CBA mice, (C) young CBA donor kid-
ney 7 days after allogeneic transplantation, (D) old CBA donor
kidney 7 days after allogeneic transplantation, (E) young CBA
donor kidney 21 days after allogeneic transplantation and (F)
old CBA donor kidney 7 days after allogeneic transplantation.
Original magnification 400×.
116
American Journal of Transplantation
2009; 9: 114–123
Donor Age, Cell Senescence and Allograft Survival
Ta b l e 1 : Basement membrane wrinkling, tubular diameter and cell number for kidneys from CBA mice
Tubular diameter Nuclei per tubular cross section (No.)
Kidney Tubular Interstitial TBM
weight (mg) atrophy (%) fibrosis wrinkling (%) All Wrinkled Normal All Wrinkled Normal
NCBA Young 236 ±54 0 0 0 1.4 ±0.08 NA 1.4 ±0.08 3.9 ±0.9 NA 3.9 ±0.9
n=20
Old 325 ±77 0 0 0 1.3 ±0.1 NA 1.3 ±0.1 4.1 ±0.8 NA 4.1 ±0.8
n=18
Allografts D7 Young 313 ±60 0 0 3.5 ±5.4 1.4 ±0.2 NA 1.4 ±0.2 4.5 ±0.7 NA 4.5 ±0.7
n=7
Old 394 ±73 12.7 ±15.5 0 60.4 ±27.91,71.0 ±0. 091,70.9 ±0.0912 1.1 ±0.12,13 3.6 ±0.523.3 ±0.712 3.9 ±0.3
n=7
Allografts D21 Young 335 ±70 31.8 ±33.5 1.0 ±0.6144.2 ±41.191.0 ±0.2 1.0 ±0.05 1.2 ±0.2 4.2 ±0.2 3.7 ±0.2 4.9 ±0.8
n=8
Old 427 ±129 43.7 ±31.8 1.1 ±0.710 63.9 ±31.370.9 ±0.0660.8 ±0. 0641.1 ±0.1 2.6 ±0.781.9 ±1.153.9 ±2.0
n=11
Isografts D7 Young 202 ±21 0 0 0 1.5 ±0.1 NA 1.5 ±0.1 5.2 ±0.4 NA 5.2 ±0.4
n=9
Old 289 ±34 3.6 ±3.3 0 33.6 ±8.03,61.2 ±0.2 1.1 ±0.2 1.3 ±0.1 4.3 ±0.5 3.8 ±0.5 4.7 ±0.7
n=7
Isografts D21 Young 189 ±12 0 0 0 1.4 ±0.1 NA 1.4 ±0.1 4.7 ±0.4 NA 4.7 ±0.4
n=4
Old 253 ±56 12.9 ±15.8 0.4 ±0.9 16.2 ±7.48,13 1.2 ±0.111 0.8 ±0.4 1.3 ±0.1 4.1 ±0.413 3.2 ±1.5 4.6 ±0.5
n=10
Data are shown for normal mice prior to transplantation (NCBA) as well as 7 days (D7) and 21 days (D21) after transplantation.
TBM =tubular basement membrane; ‘all’ =diameters or nuclei for all tubules in the 10 HPFs assessed were used to calculate the mean value for either diameter or number of
nuclei; ‘wrinkled’ =diameters or nuclei for tubules that appeared to be wrinkled in the 10 HPFs assessed were used to calculate the mean value for either diameter or number of
nuclei; ‘normal’ =diameters or nuclei for tubules that appeared to be normal in the 10 HPFs assessed were used to calculate the mean value for either diameter or number of
nuclei.
1p<0.005 when compared with young allografts D7.
2p<0.05 when compared with young allografts D7.
3p<0.05 when compared with young isografts D7.
4p<0.005 when compared with young allografts D21.
5p<0.05 when compared with young allografts D21.
6p<0.001 when compared with normal old.
7p<0.01 when compared with normal old.
8p<0.05 when compared with normal old.
9p<0.05 when compared with normal young.
10p<0.005 when compared with old allografts D7.
11p<0.01 when compared with old allografts D21.
12p<0.05 when compared with old allografts D21.
13p<0.05 when compared with old isografts D7.
American Journal of Transplantation
2009; 9: 114–123 117
Melk et al.
0
100
50
200
250
Normal
CBA D7 D21
p16
INK4a
/HPRT ratio
normalized to young normal CBA
young
old
allografts
(CBA in C57BL6)
150
p<.001
D7 D21
isografts
(CBA in CBA)
p<.05
p<.01
D7 D21
isografts
(CBA in CBA)
p<.005
n.s.
*p<.005 vs. D7 young
**p<.005 vs. D7 old
**
*
#p<.01vs. normal
old controls
#Figure 2: P16
INK4a
mRNA expres-
sion for CBA kidneys prior to and af-
ter allogeneic and syngeneic trans-
plantation. Normal CBA =prior to
transplantation; D7 =7 days after
transplantation; D21 =21 days af-
ter transplantation. Values are given
as fold difference compared with nor-
mal young CBA mice. Significant dif-
ferences are indicated.
than 0.5% of the area of cortical tubules, as expected for
age (Figure 1B). None of the old CBA mice displayed focal
and segmental glomerulosclerosis or other features of re-
nal disease. Young and old kidneys displayed no significant
differences in the tubular diameter or number of nuclei per
tubular cross-section (Table 1).
Allografts:
At day 7, allografts from old donor mice
showed greater tubular basement membrane (TBM) wrin-
kling and tubular mass loss, compared with allografts of
young donor mice (Table 1) (Figure 1C and D). TBM wrin-
kling was more frequent in old allografts (60%) compared
with young allografts (4%). Old allografts also showed
tubular atrophy (12.7% vs. 0% in young allografts), a re-
duced tubular diameter (1.0 vs. 1.4 in young D7 allografts)
and a lower number of nuclei per tubular cross-section
(3.6 vs. 4.5 in young D7 allografts) (Table 1).
At day 21, old allografts showed TBM wrinkling in 64%
of tubules and young allografts in 44% of tubules; the
difference was no longer significant (Figure 1E and F;
Table 1). Both groups manifested interstitial fibrosis when
compared with D7 allografts (old D21: p <0.005; young
D21: p <0.005). The number of tubules meeting the cri-
teria for tubular atrophy increased further in old D21 al-
lografts (44%) and became detectable in young D21 allo-
grafts transplants (32%). There was no significant differ-
ence in the amount of interstitial fibrosis or tubular atrophy
between old and young allografts at day 21. The number
of cells per tubular cross-section and the tubular diameter
further decreased in old D21 allografts (Table 1). Old D21
allografts had significantly fewer tubular cells (2.6 vs. 4.2 in
young D21 allografts; p <0.005), and the tubular diameter
tended to be smaller (0.9 vs. 1.2 in young D21 allografts;
p=0.06), particularly in tubules with wrinkled TBM
(0.8 vs. 1.0 in young D21 allografts; p <0.005).
Isografts:
The histology of young isografts at day 7 was
normal. Old isografts showed TBM wrinkling in 34% and
tubular atrophy in 4% of the tubular cross-sections. The
tubular diameter and tubular cells did not change in iso-
grafts when compared with normal young and old CBA
kidneys (Table 1).
At day 21, TBM wrinkling was not present in any young iso-
grafts, and was present in only one of the old isografts, af-
fecting 16% of the tubular cross-sections. Young isografts
did not show any tubular atrophy, whereas in old isografts,
13% of the tubular cross-sections showed tubular atrophy.
The tubular diameter and tubular cells did not change in
isografts when compared with normal young and old CBA
kidneys.
P16
INK4a
expression
Normal CBA mice:
Normal kidneys from old nor-
mal CBA expressed significantly higher p16
INK4a
mRNA
(Figure 2) compared with young kidneys (37-fold; p <
0.001). P16
INK4a
staining was assessed in the nuclei of prox-
imal and distal tubules and collecting duct (tubular staining),
podocytes and parietal epithelium of glomeruli (glomerular
staining) and interstitial cells (interstitial staining). Old nor-
mal CBA kidneys had significantly more p16
INK4a
-positive
nuclei than young normal CBA mice for tubular (p <0.005;
Figure 3A), glomerular (p <0.05; Figure 3B) and interstitial
cells (p <0.05; Figure 3C).
Allografts:
At day 7 of rejection, p16
INK4a
mRNA expres-
sion increased significantly in old allografts (p <0.01;
Figure 2) but not in young allografts (p =1.000) com-
pared with either normal old or young controls. Thus, the
rejecting old kidneys displayed higher p16
INK4a
mRNA ex-
pression than the rejecting young kidneys at day 7 (P <
0.005), reflecting both higher basal expression and greater
118
American Journal of Transplantation
2009; 9: 114–123
Donor Age, Cell Senescence and Allograft Survival
0
25
75
50
100
P16
INK4a
positive nuclei
in tubules (%)
NCBA D7 post tx D21 post tx
Old
Young
*(NCBA)
p<.005
p<.05
0
25
50
75
100
P16
INK4a
positive nuclei
in glomeruli (%)
NCBA D7 post tx D21 post tx
0
25
50
75
100
P16
INK4a
positive nuclei
in interstitium (%)
NCBA D7 post tx D21 post tx
A
B
C
p<.05
p<.05
p<.01
p<.01
*(NCBA)
*(NCBA)
*(NCBA)
*(NCBA)
*(NCBA)
*(NCBA)
*(NCBA)
Figure 3: P16
INK4a
protein expression in (A) tubular cells (B)
glomeruli and (C) interstitial cells in renal cortex of CBA kid-
neys. Normal CBA (NCBA) =prior to transplantation; D7 =7 days
after transplantation; D21 =21 days after transplantation. Values
are given as the percentage of positive nuclei for each of the three
cellular compartments. Significant differences are indicated.
induced expression. The percentage p16
INK4a
-positive nu-
clei increased by day 7: the increase was significant for
glomerular cells in old allografts (p <0.005; Figure 3B) and
for interstitial cells in both old and young allografts (p <
0.05 and p <0.001, respectively; Figure 3C). The percent-
age of p16
INK4a
-positive nuclei was significantly higher in
old than young kidneys for tubular (p <0.05), glomerular
(p <0.01) and interstitial cells (p <0.01).
At day 21, p16
INK4a
mRNA expression further increased
in allografts from old donors (p <0.005 when compared
with D7 allografts; Figure 2). In addition, p16
INK4a
mRNA
expression increased in young allografts (p <0.005 for day
21 compared with day 7; Figure 2), thus eliminating the
Normal
CBA D7 D21
allografts
(CBA in C57BL6)
0
1
2
3
4
5
N.S.
p<.01
p<.005
Ki-67 positive nuclei in tubules (%)
Figure 4: Ki-67 expression in tubular cells in renal cortex of
CBA kidneys. Normal CBA =prior to transplantation; D7 =7 days
after transplantation; D21 =21 days after transplantation. Values
are given as the percentage of positive nuclei from the total tubular
nuclei. Significant differences are indicated.
difference in p16
INK4a
mRNA expression between old and
young allografts by day 21. In all investigated cell types, the
percentage of p16
INK4a
-positive nuclei in old allografts was
significantly higher compared to normal controls (tubules:
p<0.05, glomeruli: p <0.05, interstitium: p <0.05 vs.
normal CBA; Figure 3A–C). The increase in young allografts
versus normal young kidneys was statistically significant
for glomerular and interstitial cells (p <0.05 and p <0.001
vs. normal CBA, respectively; Figure 3B and C), although
not for tubules. Thus, there was no difference in p16
INK4a
expression between old and young allografts at day 21.
Isografts at days 7 and 21:
P16
INK4a
mRNA and protein
expression for old and young isografts was similar to the
expression found in normal CBA mice (Figures 2 and 3).
Proliferation
The proliferative response of tubular cells was measured
by Ki-67 staining.
Normal CBA mice:
The rate of proliferation measurable
in tubular cells was, regardless of age, very low. Old normal
CBA kidneys had a tendency toward less Ki-67-positive
tubular cells when compared with young normal CBA mice
(Figure 4).
Allografts:
At day 7 of rejection, Ki-67 expression in-
creased significantly in old and young allografts (vs. old
American Journal of Transplantation
2009; 9: 114–123 119
Melk et al.
normal CBA: p =0.001; vs. young normal CBA: p <0.001),
but was significantly lower in old allografts compared with
young allografts (p <0.01; Figure 4). There was no further
increase in Ki-67-positive tubular cells by day 21 for either
group. The difference between transplants from old and
young donors remained significant (p <0.005).
Immune response
The immune response was assessed by histologic markers
of rejection, the amount and type of infiltrate as well as
the expression of MHC, IFN-cand cytotoxic T-cell genes
(granzyme B and perforin).
Allografts:
Both age groups developed lesions charac-
teristic of rejecting transplants at day 7 and day 21. There
were no significant differences for any of the acute features
at days 7 and 21 between young and old donor kidneys,
respectively (Table 2). The number of infiltrating cells, mea-
sured as CD45- (total lymphocytes), CD3- (T cells), CD4-
(T helper cells) and CD8- (cytotoxic T cells) positive cells,
was similar in both old and young transplants at day 7 and
day 21 (Table 3). No differences were seen for donor (CBA)
and recipient (B6) classes I and II (Table 4) expression be-
tween old and young transplants at days 7 and 21 after
transplantation. mRNA expression for two cytotoxic T-cell
genes (granzyme B and perforin) and IFN-c(Figure S1) was
similar in allografts from old and young donors at days 7
and 21 after transplantation.
Isografts:
As expected, none of the isografts from both
age groups developed lesions of rejection at day 7 or 21.
None of the kidneys showed acute tubular necrosis. One
of the old transplants showed features of mild acute tubu-
lar injury at day 7, but the others did not. There was no
interstitial infiltrate, even in the kidney with mild tubular
injury. None of the isografts at day 21 in both age groups
showed any signs of acute tubular injury.
Discussion
In the present studies, a principal difference in old donor
tissue is the earlier deterioration of old tissues with trans-
plant stress and no increase in rejection compared with
young donor tissue. Allografts from old donor kidneys
showed signs of progressive tubular atrophy and tubular
loss, whereas the number of tubular cells remained con-
sistent in young donor allografts. Transplantation also in-
duced p16
INK4a
, the cell cycle regulator typical of somatic
cell senescence, at the mRNA and protein level. The basal
levels of p16
INK4a
were present in old native donor kidneys,
and p16
INK4a
was induced in allografts from old donor kid-
neys soon after transplantation (day 7). In contrast, young
nontransplanted kidneys showed very little basal expres-
sion of p16
INK4a
, with an increased expression not until later
after transplantation (day 21). Isografts did not have an ef-
fect on p16
INK4a
expression. A proliferative response was
induced in both age groups after transplantation, but was
significantly lower in transplants from old donors. Thus, the
Ta b l e 2 : Acute histopathologic features of rejection in kidneys from CBA mice
Tubulitis in Tubulitis in Interstitial
nonatrophic tubules atrophic tubules infiltrate Glomerulitis Vasculitis Necrosis Thrombosis Edema Casts
Allografts D7 Young 0.6 ±0.5 0 2.4 ±0.5 0.7 ±0.5 0.6 ±0.5 0 0 0 0
n=7
Old 1.0 ±0.0 1.2 ±0.4 2.8 ±0.4 1.0 ±0.6 0.5 ±0.6 1.7 ±2.6 0 0 0
n=7
Allografts D21 Young 1.9 ±0.7 2.3 ±0.7 2.6 ±0.7 0.9 ±0.6 1.5 ±0.5 2.5 ±3.8 0 7.5 ±7.6 3.8 ±5.2
n=8
Old 1.1 ±0.4 1.7 ±0.5 2.7 ±0.5 1.0 ±0.0 1.6 ±0.5 1.4 ±2.4 0 7.7 ±8.4 0
n=11
Isografts D7 Young 0 0 0 0 0 0 0 0 0
n=9
Old 0 0 0 0 0 0 0 0 0
n=7
Isografts D21 Young 0 0 0 0 0 0 0 0 0
n=4
Old 0 0 0 0 0 0 0 0 0
n=10
Data are shown for mice 7 days (D7) and 21 days (D21) after transplantation.
Tubulitis, interstitial infiltrate, glomerulitis and vasculitis were assessed according to the Banff classification (28) (for details, see Materials and Methods section). The extent of graft
necrosis, thrombosis, edema and cast formation were recorded as the percentage of total parenchyma involved.
120
American Journal of Transplantation
2009; 9: 114–123
Donor Age, Cell Senescence and Allograft Survival
Ta b l e 3 : Cytology and cell counts for infiltrating lymphocytes
NCBA D7 D21
Young Old Young Old Young Old
CD45 2 ±34±8 148 ±80 138 ±86 38 ±530±7
CD3 1 ±40±1 117 ±68 111 ±79 44 ±643±10
CD4 0 ±10 47±38 54 ±61 3 ±34±3
CD8 1 ±21±2 113 ±77 88 ±67 20 ±621±8
Data are shown for normal mice prior to transplantation (NCBA)
as well as 7 days (D7) and 21 days (D21) after transplantation.
stress of transplantation induces features of cell senes-
cence in both old and young donor kidneys, and these oc-
cur earlier and to a greater extent in old donor tissues. Con-
sistent with the increase in cell senescence, we found a
reduced proliferation in response to transplantation stress.
Rejection parameters such as the Banff scores, MHC ex-
pression, cellular infiltrate and the expression of cytotoxic
T-cell genes and IFN-cwere similar in allografts from old
and young donor kidneys, both early and later after trans-
plant, suggesting that donor age does not alter immuno-
genicity. Taken together, these results show that older
donor age is an important determinant of a kidney’s re-
action to transplantation and rejection stress, but has little
effect on the magnitude of the immune response. The re-
sults establish that at least some features of aging are
induced in an accelerated fashion by stresses surrounding
transplantation.
The augmented development of histopathological features
of atrophy, cell loss and p16
INK4a
expression in isografts and
allografts from old donors indicates an increased suscep-
tibility of old allografts to damage through peri- and post-
transplant stresses. We show that acute rejection has a
greater impact in old allografts, which was not attributable
to the differences in recipient’s immune response toward
the old organ or donor’s immunogenicity. In addition, gene
array studies currently in progress in our laboratory sup-
port this finding on a much larger scale of investigated
genes. These findings are in contrast to some human data
that suggest a greater frequency of rejection and impact of
acute rejection due to higher immunogenicity of old donors
(7). The difference is that tubulitis is a late stage of epithe-
lial deterioration, with the loss of the ability to exclude
Ta b l e 4 : Donor (CBA) and recipient (B6) MHC class I and class II expression
NCBA D7 D21
Young Old Young Old Young Old
Donor MHC
Class I 0.67 ±1.40 0.14 ±0.36 1.9 ±1.8 2.0 ±1.7 0.5 ±1.4 1.8 ±1.5
Class II 0.17 ±0.38 0.08 ±0.27 2.9 ±2.0 2.2 ±1.7 0.5 ±1.4 1.5 ±1.6
Recipient MHC
Class I 0 0 3.4 ±1.1 3.5 ±0.8 2.8 ±1.0 3.5 ±0.8
Class II 0 0 2.3 ±0.5 2.2 ±1.2 3.1 ±1.1 3.7 ±0.8
Data are shown for normal mice prior to transplantation (NCBA) as well as 7 days (D7) and 21 days (D21) after transplantation. Donor
MHC expression was assessed in tubules and recipient MHC in interstitium.
lymphocytes (31). Thus, the old epithelium permits more
tubulitis with the same level of the immune response, and
the conclusion that old kidneys are more immunogeneic is
probably incorrect.
Thus, the principal problem of old donor tissues is not that
they are more immunogenic but that the parenchyma pos-
sess greater fragility, leaving it susceptible to deterioration
and atrophy and irreversible cell cycle arrest when rejec-
tion occurs. The reason for poorer outcomes in old donors
is intrinsic to the donor and is a combination of under-
lying features of somatic cell deterioration and developing
chronic features together with the loss of tubular cells. The
resulting decreased capacity of old donor tissues to repair
peritransplant injuries and maintain organ mass may be a
prototype for a general feature of old tissue. Even though
our data point toward a central role of the epithelium within
this process, we cannot exclude the possibility that older
animals may have had fewer peritubular capillaries going
into surgery, which could have led to tubular injury through
ischemia.
Not only are some mechanisms of cellular senescence
pre-established in organs from older donors but these pro-
cesses are also accelerated by the transplantation process,
in both young and old tissues. The increases in p16
INK4a
probably reflect both the response to the peri- and post-
transplant environmental stresses and the need for in-
creased replication followed by signals for cell cycle inhibi-
tion. The induction of p16
INK4a
in old donor allografts at day 7
was about 7-fold higher compared with induction in young
donor kidneys and was additive to the pre-existing higher
p16
INK4a
expression in old normal CBA mice kidney. The
changes at day 7 reflect the damage caused by acute re-
jection, and possibly by ischemia-reperfusion and stresses
of the surger y itself. It must be noted, however, that the
kidneys had minimal ischemia (as seen in the isografts), as
is the case for living donation. Since p16
INK4a
-positive cells
are irreversibly arrested in the cell cycle and are no longer
are capable of replication (17), these data suggest that, 7
days after transplantation, old donor kidneys possess a sig-
nificantly lower ability to withstand stresses and to repair,
which is supported by the Ki-67 data showing a reduced
proliferative response in old transplants. Twenty-one days
after transplantation, p16
INK4a
was increased in both old
American Journal of Transplantation
2009; 9: 114–123 121
Melk et al.
and young donor transplants, consistent with the idea that
ongoing acute rejection with increased need for replication
will exhaust the replicative potential in young donors.
The survival of an organ is limited by senescence mecha-
nisms. We believe that somatic cell senescence changes
contribute to collapse of tissue integrity and—if success-
fully bypassed—will prolong tissue survival and enhance
performance of allografts. Further studies have to investi-
gate how kidneys lacking p16
INK4a
will perform and which
cellular compartment is limiting for transplant survival.
Acknowledgments
This study was funded by Roche Organ Transplant Research Foundation,
Canadian Institutes of Health Research, Kidney Foundation of Canada, The
Muttart Foundation and The Royal Canadian Legion.
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Donor Age, Cell Senescence and Allograft Survival
Supporting Information
The following supporting information is available for this
article.
Figure S1. (A)Granzyme B, (B)perforin and (C)IFN-c
mRNA expression for CBA kidneys. Normal CBA =prior
to transplantation; D7 =7 days after transplantation; D21
=21 days after transplantation; Yg =Young. Values are
given as gene of interest/HPRT ratio. Old kidneys (•), SD
up; young kidneys (), SD down. Significant differences are
indicated.
Ta b l e S 1 : Sequences for primers and probes used in real-
time PCR studies
Please note: Wiley-Blackwell is not responsible for the con-
tent or functionality of any supporting informations sup-
plied by the authors. Any queries (other than missing ma-
terial) should be directed to the corresponding author for
the article.
American Journal of Transplantation
2009; 9: 114–123 123
