American Journal of Transplantation 2006; 6: 747–752
Blackwell Munksgaard
Subclinical Rejection Associated with Chronic
Allograft Nephropathy in Protocol Biopsies
as a Risk Factor for Late Graft Loss
F. Moresoa , M. Ibernona , M. Gomàb , M. Carrerab ,
X. Fulladosaa , M. Huesoa , S. Gil-Verneta ,
J. M. Cruzadoa , J. Torrasa , J. M. Grinyóa
and D. Seróna, ∗
a cidence of SCR is rather variable between centers and is
Nephrology and b Pathology Departments, Hospital
Universitari de Bellvitge, IDIBELL (Institut d’Investigació
de Bellvitge), C/Feixa Llarga s/n, L’Hospitalet 08907
Barcelona, Spain
∗ Corresponding author: Daniel Serón,
17664dsm@comb.es
Chronic allograft nephropathy (CAN) in protocol biop-
sies is associated with graft loss while the association
between subclinical rejection (SCR) and outcome has
yielded contradictory results. We analyze the predic-
tive value of SCR and/or CAN in protocol biopsies on
death-censored graft survival. Since 1988, a protocol
biopsy was done during the first 6 months in stable
grafts with serum creatinine <300 lmol/L and protein-
uria <1 g/day. Biopsies were evaluated according to
Banff criteria. Borderline changes and acute rejection
were grouped as SCR. CAN was defined as presence of
interstitial fibrosis and tubular atrophy. Mean follow-
up was 91 ± 46 months. Sufficient tissue was obtained
in 435 transplants. Biopsies were classified as normal
(n = 186), SCR (n = 74), CAN (n = 110) and SCR with
CAN (n = 65). Presence of SCR with CAN was asso-
ciated with old donors, percentage of panel reactive
antibodies and presence of acute rejection before pro-
tocol biopsy. Cox regression analysis showed that SCR
with CAN (relative risk [RR]: 1.86, 95% confidence in-
terval [CI]: 1.11–3.12; p = 0.02) and hepatitis C virus
(RR: 2.27, 95% CI: 1.38–3.75; p = 0.01) were indepen-
dent predictors of graft survival. In protocol biopsies,
the detrimental effect of interstitial fibrosis/tubular
atrophy on long-term graft survival is modulated by
SCR.
Key words: Chronic allograft nephropathy, protocol
biopsies, renal transplantation, subclinical rejection
Received 15 September 2005, revised 7 November 2005
and accepted for publication 28 November 2005
Introduction
Different centers have conducted prospective studies of
protocol biopsies in order to characterize histological le-
C 2006 The Authors
Journal compilation 
C 2006 The American Society of
Transplantation and the American Society of Transplant Surgeons
doi: 10.1111/j.1600-6143.2005.01230.x
sions in renal allografts with otherwise stable renal function
(1–5). There is agreement that subclinical rejection (SCR),
that is, the presence of histological lesions of rejection in
well functioning grafts, peaks during the initial months of
post-transplantation and declines thereafter (6,7). The in-
influenced by the timing of protocol biopsy, the presence
of an episode of acute rejection before the protocol biopsy
and immunosuppressive treatment (6,8–10). The potential
influence of other variables on the incidence of SCR such
as donor source (deceased vs. living) or recipient type (pe-
diatric vs adult) has not been properly evaluated (11,12).
In protocol biopsies, chronic allograft nephropathy (CAN) is
a frequent finding that progresses rapidly during the first
year and slower but steadily thereafter (4,13). The pres-
ence of CAN is associated with different clinical variables
such as donor age, an episode of acute rejection before
biopsy, the use of calcineurin inhibitors or timing of pro-
tocol biopsy (2,3). The presence of CAN in protocol biop-
sies has been consistently associated with a decreased
long-term renal allograft survival. Furthermore, it has been
shown that CAN is an independent predictor of graft failure
from renal function and proteinuria, suggesting that proto-
col biopsies contain useful information to predict outcome
that is not present in clinical data (2,3,8).
In studies of serial protocol biopsies, the presence of SCR
precedes the appearance of CAN suggesting that SCR
should be also associated with a poorer long-term out-
come (6,14). However, studies evaluating the predictive
value of SCR on long-term graft survival have yielded con-
tradictory results (8,11,14,15). Thus, in the present study
we analyzed long-term-death-censored graft survival in a
large cohort of deceased donor renal transplants with a
protocol biopsy taking into consideration the presence of
SCR, CAN or the association of both diagnosis in the same
biopsy.
Patients and Methods
Study design
Since June 1988, a prospective protocol renal allograft biopsy is performed
in patients who give their informed consent and fulfill the following criteria:
(a) serum creatinine <300 lmol/L; (b) proteinuria <1g/24 h and (c) stable
renal function, defined as variability of serum creatinine of less than 15%
during 2 weeks before and after biopsy.
747
Moreso et al.

The timing of protocol biopsy has been progressively delayed in order to Table 1: Demographic characteristics of patients and clinical evo-
better focus on the study of early chronic lesions. Between 1988 and 1990 lution after transplantation
the biopsy was done during the 1st month while later on the biopsy was Variable Mean ± SD Range
done between the 3rd and 6th month. For the present study, we considered
patients biopsied until December 2003. Donor age (years) 38 ± 17 (6–77)
Donor gender (male/female) 302/133
Recipient age (years) 46 ± 14 (11–76)
Biopsies Recipı̀ent gender (male/female) 279/156
Biopsies were performed under ultrasound guidance with an automated Etiology of ESRD
gun, processed for routine light microscopy and stained with hematoxilin- Glomerular 178
eosine, periodic acid Schiff, Masson’s trichrome and silver-methenamine. Insterstitial 85
Renal lesions in protocol biopsies were blindly graded and diagnosed accord- APKD 56
ing to the 1997 Banff criteria (16) in the absence of any clinical information. Nephrosclerosis 26
Only biopsies containing at least one arterial section and one glomerular Diabetes 4
profile were considered. SCR was defined as the presence of i-score ≥1 Unknown 86
and t-score ≥1. The presence of CAN was defined as ci+ct scores ≥ 2. PRA (%) 8 ± 19 (0–100)
HCV status (negative/positive) 366/69
Protocol biopsies were not considered in the clinical management of HLA mismatches 3.0 ± 1.1 (0–5)
patients. Cold ischemia time (h) 22 ± 6 (7–49)
Delayed graft function (no/yes) 358/77
Clinical variables Acute rejection (no/yes) 345/90
The following variables were evaluated in each patient at the time of surgery:
ESRD = end-stage renal disease; APKD = adult polycystic kidney
age and gender of the donor and the recipient, number of transplant, pres-
disease; PRA = panel reactive antibodies; HCV = hepatitis C
ence of hepatitis C virus antibodies, last panel reactive antibodies, number
virus; HLA = human leukocyte antigen.
of HLA mismatches, cold ischemia time and immunosuppressive therapy.

After surgery, the presence of delayed graft function and acute rejection
were evaluated. At the time of protocol biopsies and during follow-up serum
creatinine, proteinuria, total serum cholesterol, CsA or TAC dose and levels
were recorded.
Patient and death-censored graft survival were evaluated on December
2004.
Statistics
Results are expressed as the mean ± standard deviation. Comparison be-
tween groups was performed by means of chi-square test for categori-
cal data, Kruskal-Wallis test for ordinal data and analysis of variance with
Scheffé post hoc test for normally distributed continuous variables. Death-
censored graft survival was considered the outcome variable in the present
study. Graft survival was estimated by the Kaplan-Meier method and com-
parison between groups was done by the log-rank test. Univariate and mul-
tivariate Cox regression analysis were employed to analyzed variables as-
sociated with death-censored graft survival. All p values were 2-tailed and
a p-value < 0.05 was considered significant.
Results
Patients
During the study period, 472 protocol biopsies were
done in 463 patients. In 37 cases (7.8%), an insufficient
sample for histological evaluation was obtained. Accord-
ingly, 435 renal transplants were included in the present
study. Clinical characteristics of patients are summarized in
Table 1. Immunosuppressive treatment received at the
time of transplantation was as follows: cyclosporine (CsA)
and prednisone (n = 23); CsA and prednisone associated
with polyclonal or monoclonal anti-lymphocytic antibodies
(n = 171); CsA, azathioprine and steroids (n = 35); CsA,
mycophenolate mofetil and steroids (n = 124); tacrolimus,
mycophenolate mofetil and steroids (n = 49); a calcineurin-
free regimen based on anti-lymphocytic antibodies, my-
cophenolate mofetil and steroids (n = 23); CsA, sirolimus
and steroids (n = 10).
At the end of follow-up 31 patients died with a function-
ing graft and 81 grafts were lost. Causes of patient’s death
were cardiovascular disease (n = 15), neoplasia (n = 11),
infection (n = 3) and chronic liver failure (n = 2). Causes
of graft loss were CAN (n = 63), hepatitis C virus asso-
ciated glomerulonephritis (n = 7), de novo membranous
glomerulonephritis (n = 4), recurrence of primary glomeru-
lonephritis (n = 3), nontreatment compliance (n = 3) and
transplantectomy due to Kaposi’s sarcoma involving the
graft hilium (n = 1). Mean follow-up was 91 ± 46 months.
Biopsies
According to Banff criteria 265 biopsies were adequate
(at least 10 glomerular and 2 arterial sections), 89 were
marginal (between 7 and 9 glomerular and at least 1 ar-
terial section) and 81 were insufficient (between 1 and
6 glomerular sections and at least 1 arterial section).
Marginal samples contained 8 ± 1 and insufficient sam-
ples 4 ± 1 glomerular sections, respectively. Clinical data
at the time of protocol biopsy are summarized in Table 2.
Table 2: Clinical data at the time of protocol biopsy
Variable Mean ± SD Range
Time of biopsy (days) 120 ± 44 (13–210)
Serum creatinine (lmol/L) 142 ± 48 (63–300)
Proteinuria (g/day) 0.31 ± 0.22 (0.02–1.00)
Mean arterial pressure (mmHg) 102 ± 12 (67–141)

748 American Journal of Transplantation 2006; 6: 747–752

 Subclinical Rejection and Graft Outcome

Histological diagnosis according to Banff criteria were nor-

mal (n = 186, 43%), SCR (n = 74, 17%), CAN (n = 110,
25%) and CAN associated with SCR (n = 65, 15%). The
incidence of CAN in patients with SCR was 47% and in
patients without SCR was 37%, p = 0.05).

The severity of SCR was not different in biopsies with or

without CAN (n = 49 (75.4%), n = 13 (20.0%) and n = 3
(4.6%) versus n = 61 (82.4%), n = 11 (14.8%) and n = 2
(2.7%) for borderline changes, acute rejection type I and II,
respectively). The severity of CAN was also not different
in biopsies displaying CAN with or without SCR (n = 52
(80.0%), n = 12 (18.5%) and n = 1 (1.5%) vs. n = 88
(80.0%), n = 21 (19.0%) and n = 1 (1.0%) for CAN type I,
II and III, respectively). Similarly, the severity of cg and cv
scores was not significantly different in patients displaying
CAN with or without SCR (0.24 ± 0.43 vs. 0.35 ± 0.48 for
cg and 0.45 ± 0.77 vs. 0.43 ± 0.77 for cv).
Clinical characteristics of patients according to
histological diagnosis
Demographic characteristics and clinical evolution of pa-
tients according to histological diagnosis are summarized
in Table 3. Patients with CAN received kidneys from older
donors, the number of acute rejection episodes was higher
in patients with SCR and the degree of sensitation was
the highest in patients with CAN and SCR in the protocol
biopsy. Only two patients were treated for a clinical episode
of acute rejection after protocol biopsy.
The incidence of SCR was also associated with immuno-
suppressive treatment. Patients receiving a tacrolimus-
based regimen showed the lowest incidence of SCR
(16%), while patients receiving a calcineurin-free regimen
showed the highest incidence (56%). On the other hand,
the incidence of CAN was not statistically different in pa-
tients treated with CsA, tacrolimus or a calcineurin-free
regimen (40%, 43% and 30%, respectively) (Figure 1).
At the time of protocol biopsy, patients with CAN asso-
ciated with SCR showed the poorest graft function and
Figure 1: Percentage of patients with different histological
diagnosis in the protocol biopsy according to immunosup-
pressive treatment. SCR, subclinical rejection; CAN, chronic allo-
graft nephropathy; CsA, cyclosporine; TAC, tacrolimus; CNI-free,
calcineurin inhibitor free immunosuppresion.
the highest degree of proteinuria (Table 4). As shown in
Table 4, trough CsA or tacrolimus plasma levels at the
time of biopsy were not different in the four groups of
patients.
Death-censored graft survival and histological
diagnosis
Death-censored graft survival was lower in patients with
SCR associated with CAN than in the other groups
(Figure 2). Death-censored graft survival in patients with
borderline acute rejection and patients with acute rejection
was not different.
Univariate and multivariate Cox regression analysis was
performed to analyze clinical, analytical and histological
variables associated with death-censored graft survival.
For this analysis, continuous variables were binarized
according to the mean and all regressions were done

Table 3: Characteristics of patients according to histological diagnosis

Variable Normal SCR CAN CAN + SCR P
N 186 74 110 65
Donor age (years) 36 ± 16 35 ± 19 40 ± 161,2 41 ± 171,2 0.032
Donor gender (m/f) 122/64 52/22 81/29 48/17 Ns
Recipient age (years) 45 ± 13 45 ± 16 45 ± 14 46 ± 13 Ns
Recipient gender (m/f) 117/69 49/25 74/36 39/26 Ns
PRA (%) 7 ± 20 8 ± 22 5 ± 13 14 ± 231,3 0.032
HCV (negative/positive) 152/34 65/9 93/17 56/9 ns
HLA mismatches 3.0 ± 1.1 2.9 ± 1.0 3.0 ± 1.1 3.1 ± 0.9 ns
CIT (h) 22 ± 6 22 ± 6 23 ± 6 24 ± 7 ns
DGF (%) 17 12 18 26 ns
Acute rejection (%) 12 28 21 35 <0.001
SCR = subclinical rejection; CAN = chronic allograft nephropathy; m = male; f = female; PRA = panel reactive antibodies;
HCV = hepatitis C virus; HLA = human leukocyte antigens; CIT = cold ischemia time; DGF = delayed graft function.
1 p < 0.05 versus normal group, 2 p < 0.05 versus SCR group, 3 p < 0.05 versus CAN group.

American Journal of Transplantation 2006; 6: 747–752 749

Moreso et al.

Table 4: Clinical data at the time of protocol biopsy according to histological diagnosis
Variable Normal SCR CAN CAN+SCR p
N 186 74 110 65
SCr (lmol/L) 132 ± 42 136 ± 40 150 ± 501,2 166 ± 551,2,3 <0.001
Proteinuria (g/day) 0.27 ± 0.19 0.31 ± 0.21 0.33 ± 0.241 0.42 ± 0.271,2,3 <0.001
MAP (mmHg) 99 ± 12 102 ± 12 104 ± 121 104 ± 121 0.005
CsA levels 192 ± 76 (155) 192 ± 70 (59) 209 ± 82 (90) 200 ± 92 (58) ns
TAC levels 9 ± 3 (24) 10 ± 4 (5) 10 ± 4 (20) 7 ± 4 (4) ns
SCR = subclinical rejection; CAN = chronic allograft nephropathy; SCr = serum creatinine; MAP = mean arterial
pressure; CsA = cyclosporine; TAC = tacrolimus. 1 p < 0.05 versus normal group, 2 p < 0.05 versus SCR group,
3 p < 0.05 versus CAN group. In the case of cyclosporine and tacrolimus levels the number of patients is shown between

brackets.

The association between histological diagnosis and graft

loss was confirmed considering only hepatitis C virus neg-
ative patients and graft loss due to CAN as the outcome
variable (relative risk [RR]: 2.04, 95% confidence inter-
val [CI]: 1.02–4.08; p = 0.04). Similarly, the association
between histological diagnosis and death-censored graft
survival was also confirmed when data were reanalyzed
only considering the 331 patients with serum creatinine
<200 lmol/L at the time of biopsy and a biopsy contain-
ing at least 7 glomeruli (RR: 1.97, 95% CI: 1.02–3.82; p =
0.04).

Discussion

Figure 2: Death censored graft survival according to histolog-
ical diagnosis.
adjusting for the timing of protocol biopsy. In Table 5,
variables associated with death-censored graft survival in
the univariate Cox regression analysis are summarized.
Neither immunosuppressive treatment nor acute rejection
episodes before the protocol biopsy were associated with
graft survival. Finally, multivariate Cox regression analysis
showed that only the presence of SCR associated with
CAN in the protocol biopsy and hepatitis C virus were
independent predictors of death-censored graft survival
(Table 5).
In the present study, we observed that death-censored
long-term graft survival was significantly decreased in pa-
tients simultaneously displaying SCR and CAN in the proto-
col biopsy. Graft survival in patients displaying SCR or CAN
was not different from patients with a normal biopsy. Fur-
thermore, intermediate outcome variables such as serum
creatinine, mean arterial pressure or proteinuria at the time
of biopsy were higher in patients simultaneously display-
ing SCR and CAN. This result suggests that the detrimental
effect of CAN on outcome is modulated by the presence
of SCR.
The criteria to define SCR in different studies have not been
homogeneous. Some authors have arbitrarily defined SCR

Table 5: Variables associated with death censored graft survival by univariate and multivariate Cox regression analysis
adjusting for the timing of the protocol biopsy
Univariate analysis Multivariate analysis
Variable Category RR CI 95% P RR CI 95% p
Donor age ≥38 years 1.92 1.21–3.03 <0.01 1.47 0.90–2.42 0.12
PRA ≥8% 1.89 1.19–3.02 <0.01 1.51 0.92–2.46 0.10
HCV Positive 2.52 1.58–4.03 <0.01 2.27 1.38–3.75 <0.01
SCr ≥140 lmol/L 2.14 1.37–3.33 <0.01 1.59 0.98–2.59 0.06
Proteinuria ≥0.31 g/day 2.03 1.30–3.17 <0.01 1.46 0.92–2.32 0.11
Histology CAN + SCR 2.03 1.24–3.35 <0.01 1.86 1.11–3.12 0.02
RR = relative risk; CI 95% = confidence interval of 95%; PRA = panel reactive antibodies; HCV = hepatitis C virus; SCr =
serum creatinine at the time of protocol biopsy; CAN + SCR = chronic allograft nephropathy with subclinical rejection in
the protocol biopsy.

750 American Journal of Transplantation 2006; 6: 747–752


as the presence of at least acute rejection grade Ia while
others have also included patients with borderline changes
or even nonspecific inflammatory changes (1–4,11,17). The
difference between borderline changes and acute rejection
depends on the number of inflammatory cells in the tubular
epithelium (16), but there can be some overlap between
both diagnosis, since the evaluation of tubulitis is asso-
ciated with a significant inter-observer variability (18,19).
In studies evaluating the predictive value of biopsies per-
formed in clinical episodes of acute rejection, outcome in
patients with borderline changes and acute rejection grade
I is not different (20). Moreover, in protocol biopsy stud-
ies there is controversy whether borderline changes and
acute rejection imply a different prognosis (6,8,11,15,17).
In the present study, three-quarters of patients with SCR
displayed borderline changes in agreement with previous
studies. Furthermore, graft survival was not different in pa-
tients with borderline changes and acute rejection. Thus,
for the present study, both groups were considered as
SCR.
The presence of CAN in protocol biopsies has been re-
peatedly associated with decreased renal allograft survival
(2–4). Nevertheless, studies evaluating the predictive value
of SCR on renal allograft outcome are not conclusive. In a
prospective randomized study performed in a single cen-
ter, it has been shown that treatment of SCR evaluated by
means of early protocol biopsies with steroids pulses is
associated with a lower chronic damage score in 6-month
protocol biopsies and with a lower 2-year serum creatinine
(14). The sequential association between SCR and CAN
has been confirmed in a large study of serial protocol biop-
sies (4,6). In the present study, we observed an association
between SCR and CAN in the protocol biopsy, further sug-
gesting a link between both entities.
In our study, patients with SCR without CAN as well as
patients with CAN without SCR had an outcome that was
not different from patients with a normal protocol biopsy,
suggesting that neither SCR nor CAN diagnosed at an early
stage are major determinants of graft survival. On the con-
trary, graft survival was lower in patients with SCR and
CAN. Recently, Cosio et al. have observed that patients
with SCR and CAN fair less well than patients only display-
ing CAN in a 1 year protocol biopsy study (21). However,
in their study the number of patients only displaying SCR
was too low for statistical analysis. Thus, our data suggest
that the presence of persistent inflammation in kidneys al-
ready displaying chronic lesions is associated with a higher
probability for progression to end-stage renal disease. Our
data do not allow to further explore mechanisms leading
to graft loss in patients with SCR and CAN, but two dif-
ferent mechanisms of renal damage may fit this observa-
tion. In one hand, it could be proposed that patients with
SCR and CAN have a more severe inflammation than pa-
tients with SCR without CAN. However, the severity of
acute lesions evaluated according to Banff criteria in both
groups was not different. Unfortunately, we have not stud-
American Journal of Transplantation 2006; 6: 747–752
Subclinical Rejection and Graft Outcome
ied the phenotype of infiltrating cells or cytokine profile
in order to explore qualitative differences in inflammatory
cells. Alternatively, it could be argued that kidneys with
CAN have a decreased capacity for tissue repair in front of
different insults including persistent inflammation (22). In
our study, patients with SCR and CAN showed the high-
est degree of sensitation and suffered from the highest
incidence of acute rejection episodes before the protocol
biopsy. This association favors the idea that alloimmune
response maybe more intense in this group of patients.
On the other hand, patients with CAN, either with or with-
out SCR, received kidneys from older donors which favors
that kidneys with CAN may be more susceptible to in-
flammatory mediated damage. Taken together, our results
suggest that both mechanisms, enhanced alloimmune re-
sponse and impaired tissue repair capacity, may contribute
to decreased graft survival in patients displaying SCR with
CAN. Regardless of the mechanism leading to graft loss
in these patients, an epidemiological consequence of this
observation is that previous studies evaluating the predic-
tive value of CAN on outcome may have overestimated its
impact while studies evaluating SCR may have underesti-
mated its relevance.
Finally, our data raise the question whether patients dis-
playing SCR with or without CAN could benefit from a spe-
cific treatment. Rush et al. showed that treatment of SCR
with steroid pulses may slow the progression of chronic le-
sions (14). Moreover, tacrolimus therapy is associated with
a reduced incidence of SCR, specially when it is associated
with MMF (4,9,10). These data suggest that SCR can be
treated with increased immunosuppression. However, in
the present study we did not observe a negative impact of
SCR without CAN on long-term graft survival. Accordingly,
the potential benefit of increasing immunosuppression in
these patients is doubtful and may be counterbalanced by
adverse events associated with overimmunosuppression
(23). On the contrary, our data suggest that patients dis-
playing SCR with CAN are the most interesting group to be
considered in a trial aimed to prevent progression of CAN.
Recently, it has been shown that treatment with steroid
pulses has no benefit in patients displaying SCR with CAN
in serial protocol biopsies done from the first year (12).
Thus, clinical prospective trials will be necessary to evalu-
ate whether these patients may benefit of increasing non-
nephrotoxic immunosuppression (24–26).
In summary, our data suggest that in protocol biop-
sies the detrimental effect of interstitial fibrosis/tubular
atrophy on long-term graft survival is modulated by
SCR.
Acknowledgments
This work was supported by FIS grants to F. Moreso (PI 04/0177) and D.
Seron (PI 04/0086). M. Ibernon was supported by an IDIBELL grant and M
Gomà by an Astellas Spain Grant.
751
Moreso et al.
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