Subclinical Rejection: The Sword of Damocles in Renal Transplantation?
John Lyons, PharmD
PGY-2 Solid Organ Transplant Pharmacy Resident
University Health System, San Antonio, TX
Division of Pharmacotherapy, The University of Texas at Austin College of Pharmacy
Pharmacotherapy Education and Research Center
University of Texas Health Science Center at San Antonio
January 13th
, 2016
Learning Objectives:
1. Describe the pathophysiology of renal transplant rejection
2. Define subclinical rejection and the role of the protocol biopsy in renal transplantation
3. Review current literature regarding the utility of treatment of subclinical rejection in renal
transplant recipients
4. Develop an evidence-based recommendation regarding the treatment of subclinical rejection
in renal transplant recipients
Lyons | { PAGE }
RENAL TRANSPLANTATION AND MAINTENANCE IMMUNOSUPPRESSION1-6
A. Renal transplantation1
a. Treatment of choice for end stage renal disease
b. Substantial benefit in comparison to long-term dialysis
i. Mortality
ii. Quality of life
iii. Cost
B. Goals of immunosuppression1,2
a. Prevent acute rejection of allograft
b. Prevent early graft loss
C. 1-year graft rejection has decreased and graft survival has increased steadily since the
1960s, correlating with development and release of new immunosuppressive agents2-6
Figure 1. 1-year graft survival and rejection rates, 1960-20133
D. Despite advances in immunosuppression and an impressive decrease in rejection rates
within the first year post-transplant, long-term renal allograft survival has not
significantly benefited3
a. Graft failure still remains roughly 4% per year
b. Chronic rejection is the second leading cause of graft failure
c. Acute rejection is an infrequent cause of graft failure
d. Epidemiologic studies have found a strong correlation between acute rejection
episodes and development of chronic allograft damage and allograft failure
0
10
20
30
40
50
60
70
80
90
100
' 6 5 ' 7 0 ' 7 5 ' 8 0 ' 8 3 ' 8 5 ' 9 4 ' 9 5 ' 9 6 ' 9 8 ' 9 9 ' 0 0 ' 0 4 ' 0 8 ' 1 0 ' 1 2
1 year graft survival 1 year rejection rate
Lyons | { PAGE }
e. Number, severity, and type of acute rejection episodes are strongly correlated
with early graft failure
Figure 2. Long-term Renal Transplant Graft Survival3
RENAL ALLOGRAFT REJECTION1-2, 7-9
A. Graft rejection may be cellular and/or humoral1-2,, 7-9
a. Although both pathways are intertwined, damage to the graft is dependent on cell
pathway orchestrating the immune response
B. Cellular graft rejection – “three signal model” of alloimmune responses1,2,9
a. Antigen-presenting cells (APC) identify non-self in graft and process graft antigen
for presentation
i. Engage alloantigen-reactive T-cells and memory T-cells
b. Signal 1
i. Antigen processed and presented by APC triggers T-cell activation
through T-cell receptor
c. Signal 2
i. Also known as “co-stimulation”
ii. Combination of signal 1 and 2 triggers signal transduction pathways that
lead to production of important inflammatory molecules
1. Interleukin-2
2. Tumor necrosis factor alpha
iii. These molecules play a role in activation of signal 3
d. Signal 3
i. Trigger for T-cell proliferation
ii. Leads to
1. Nucleotide synthesis
2. Differentiation of T-cells into various effector T-cells
e. Effector T-cells and graft damage
i. Effector T-cells emerge from lymphoid organs and orchestrate
inflammatory response
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12
Gra
ft S
urv
ival
Years Post Transplant
Lyons | { PAGE }
ii. Infiltrate graft
iii. Activates several cell lines, including macrophages, B cells, plasma cells,
and chemokines
iv. Typical graft damage involves
1. Infiltration of the kidney tubules by mononuclear cells and intima
of small arteries
2. Direct cell lysis from cytotoxic T-cells and activated effector cells
C. Humoral graft rejection1,7-9
a. Also commonly known as antibody-mediated rejection (AMR)
b. Antigen presented to B cells, which differentiate and mature into active antibody-
producing plasma cells
c. Antibodies produced by plasma cells mark cells for destruction
i. Activates complement cascade
1. Binding of C1q to antigen-antibody complex on graft endothelium
triggers activation of complement cascade
2. End-product is membrane attack complex (MAC) which attacks
and destroys integrity of phospholipid bilayer of cells leading to
cell death
ii. Forms immune complexes which target cell for destruction via
macrophages or natural killer cells
d. Transplant recipients may develop donor-specific antibodies (DSA) to donor
human leukocyte antigen (HLA)
D. Types of rejection in renal transplantation1-2
a. Hyperacute rejection
i. Occurs when recipient’s immune system immediately rejects the donor
organ
ii. Typically occurs upon reperfusion of the newly transplanted organ, but
can be delayed up to 3 days post-transplant
iii. Characterized by rapid, widespread vascular thrombosis affecting arteries,
arterioles, and glomeruli
iv. With the development of detailed HLA phenotyping techniques and
donor-recipient cross-matching, incidence of hyperacute rejection is rare
b. Acute rejection
i. Most commonly refers to acute cellular rejection (ACR)
ii. Can occur at any time post-transplant
iii. 2 predominant forms
1. Cellular rejection (most common)
2. Humoral rejection
iv. Cellular rejection
1. Most common form of acute rejection
2. T-cell-mediated
3. Inflammatory infiltrates typically diminish rapidly upon successful
treatment, but edema, tubular inflammation, and tubular cell
damage may persist
v. Acute humoral rejection
1. Mediated by antibody-producing plasma cells
Lyons | { PAGE }
2. Diverse histologic appearance
3. Characterized by diffuse peritubular capillary staining for
complement component C4d, although not necessary
a. Linked to presence of DSAs and humoral rejection
CHRONIC REJECTION1, 10-12
A. Terminology and definition1, 10-12
a. Previously referred to as chronic allograft nephropathy (CAN)1
b. Due to presence of both alloimmune and non-alloimmune mechanism of
progressive graft injury, the terms “chronic rejection” and “chronic allograft
nephropathy” have been largely replaced by “interstitial fibrosis and tubular
atrophy” (IFTA)1
i. Now commonly referred to as CAN/IFTA
c. Progressive decline in allograft function gradually leading to allograft failure10-12
i. Multi-factorial
ii. Involves immunologic as well as non-immunologic factors
1. Immunologic
a. Cellular rejection
b. Humoral rejection
c. Medication non-adherence
2. Non-immunologic
a. Delayed graft function (DGF)
b. Infection
c. Hypertension
d. Post-transplant diabetes mellitus
e. Calcineurin inhibitor (CNI) toxicity
iii. Second leading cause of graft loss
d. Characterized by1, 11
i. Patch interstitial fibrosis with infiltrates of lymphocytes, plasma cells, and
mast cells
ii. Tubular atrophy or tubular dropout
iii. Arterial wall thickening with intimal fibrosis
iv. Glomeruli are often abnormal, constituting a lesion known as chronic
transplant glomerulopathy
Lyons | 6
Table 1. Risk Factors for the Development of CAN/IFTA10,12
Donor Derived Recipient Derived Deceased donor kidney
Non-heart beating donor kidney
Donor age > 60
Female Donor
Donor with prior cardiac history or vascular disease
Cold ischemic time
DGF
Obesity
Polyomavirus nephropathy
CNI toxicity
Recurrent renal disease or de novo glomerulopathy
Hypertension
Hyperlipidemia
Proteinuria
Diabetes
Medication non-compliance
HLA mismatch
Recipient pre-sensitization/panel reactive antibody
(PRA)
Presence of donor specific antibody (DSA)
Acute rejection
Subclinical rejection
TREATMENT OF REJECTION7-8, 10,13
A. KDIGO Guideline for the Care of Kidney Transplant Recipients: Treatment for Rejection
(2009)13
a. Cellular Rejection
i. First line, mild-moderate: High-dose or “pulse-dose” corticosteroids
ii. Steroid-resistant rejection or severe rejection: Rabbit antithymocyte
globulin
b. Humoral Rejection7-8, 13
i. Treatment is targeted at removing circulating DSA, B-cells, and antibody-
producing plasma cells
ii. Plasmapheresis and intravenous immune globulin (IVIG) serve as the
backbone for antibody-mediated rejection treatment
1. Rapidly removes circulating DSA
2. IVIG replaces IgG that is removed during the plasmapheresis
process, and is believed to have an effect on decreasing and
preventing rebound DSA production
iii. Targeted antibody therapies can be used in conjunction with
plasmapheresis and IVIG, with or without corticosteroids
1. Rituximab
2. Bortezomib (not included in 2009 KDIGO Guidelines)
3. Eculizumab (not included in 2009 KDIGO Guidelines)
c. Chronic rejection10
i. No specific treatment modality has been identified
ii. The best “treatment” for chronic rejection/IFTA is prevention
1. Prompt diagnosis and treatment of acute rejection episodes
Lyons | { PAGE }
2. Management of post-transplantation hypertension, hyperlipidemia,
diabetes, and proteinuria
3. Optimization of immunosuppressive medications, especially CNIs
4. Maintenance of medication adherence
THE PROTOCOL BIOPSY14-25
A. Definition14-23
a. Biopsy performed at pre-specified post-transplant milestones regardless of
allograft function
b. Surveillance in nature
c. Performed in less than 20% of transplant centers in the U.S.26
B. Serum creatinine: an old friend that’s not keeping up24-25
a. Serum creatinine and proteinuria lack sensitivity as measurement for kidney
dysfunction24
i. Although widely used due to its ease of measurement and relatively low
cost, using serum creatinine as measure of glomerular filtration rate (GFR)
has several disadvantages
ii. Serum creatinine has wide inter-patient variability with regard to
1. Sex
2. Age
3. Race
4. Muscle mass and body weight
iii. Change in serum creatinine may not manifest until underlying pathology is
advanced
1. Wide inter-assay variability exists between labs25
b. Entities such as early acute rejection and recurrent focal segmental
glomerulosclerosis develop within hours to days post-transplant and become
clinically apparent early26
i. All will present as an increase in serum creatinine
ii. Using serum creatinine as primary marker lacks specificity for the
pathologic etiology
c. Other causes of graft dysfunction develop over a longer period of time26,27
i. Can be preceded by a “subclinical phase”
ii. Changes and damage to graft can elude routine laboratory monitoring
d. Other markers, with greater sensitivity and specificity for monitoring graft
function, have been proposed24,26
i. Urinary biomarkers such as cystatin c
ii. Serial 24-hour urine creatinine collections and GFR monitoring
iii. Protocol biopsies
iv. None of these methods are without limitations
C. Advantages of protocol biopsies14-23
a. Detect incidental findings
i. BK virus nephropathy
ii. CNI toxicity
iii. Chronic rejection
iv. Primary disease recurrence
Lyons | { PAGE }
v. De novo glomerulonephritis
vi. Asymptomatic urinary tract infections
b. Identify early clinical rejection
c. Identify subclinical rejection (SubR)
d. Provide a more useful marker than serum creatinine alone in monitoring graft
function and changes over time in high immunologic risk renal transplant
recipients
i. Identification of early subclinical rejection on protocol biopsy has allowed
for the opportunity for prompt treatment and modification of maintenance
immunosuppression to prevention of progression28
D. Disadvantages of protocol biopsies14-23
a. Invasive
b. Risk of complications
i. Hematuria
ii. Perinephric hematoma
iii. Bowel perforation
iv. Vasovagal reaction
v. Graft loss
c. Sampling error
d. Inter- and intra-observer variability among pathologists
e. Cost
f. Patient quality of life
SUBCLINICAL REJECTION26-46
A. Definition/Epidemiology26-27, 29-33
a. Presence of histological features of acute rejection on renal biopsy in absence of a
decline in renal function
b. Clinically significant decline in renal function - increase in serum creatinine ≥ 10-
25% of baseline29
c. Prevalence is highest early post-transplant
i. Most common in first 6 months post-transplant
ii. One of the earliest studies evaluating subclinical rejection reported an
incidence of 30% within the first 3 months of transplant in the era of
cyclosporine (CsA)-based immunosuppression32
iii. Declines to 18% at 12 months post-transplant
iv. Incidence of subclinical rejection in the era of tacrolimus (TAC)-based
immunosuppression has declined tremendously
1. 6-8% within 1-3 months post-transplant33
B. Pathophysiology26, 34-36
a. Alloimmune process similar to clinical acute rejection
b. Key differences and similarities between subclinical and clinical rejection
i. Macrophage activation marker allograft inflammatory factor-1 found to be
10-fold higher in patients with clinical rejection
1. Detrimental effect on renal function in clinical rejection may be
due to a vasoconstrictive or cytotoxic substances produced by
macrophages
Lyons | { PAGE }
ii. Reduced chemokine, cytokine, and cytotoxic lymphocyte product amounts
in comparison to clinical rejection
C. Risk factors37-39
a. HLA mismatch
i. Class II HLA antigen mismatch (DR, DP, DQ) more closely associated
with subclinical rejection in comparison to Class I HLA antigen mismatch
(A, B, C)
b. Prior sensitization
i. Blood transfusion
ii. Pregnancy
iii. Previous organ transplant
iv. Infection
c. Previous clinical rejection
d. Induction immunosuppression with interleukin-2 receptor antagonists (IL-2RA)39
D. Correlation to acute and chronic rejection35, 40
a. Major clinical concern surrounding subclinical rejection pertains to potential
implications on long-term graft damage and survival
i. Previous literature demonstrated that a cohort of patients with a 7-day
post-transplant biopsy showing subclinical rejection go on to develop
clinical rejection if untreated40
ii. Of patients with borderline changes on surveillance biopsy, 20%
experienced acute rejection within 6 months in a single-center study40
iii. Subclinical rejection found on 3-month protocol biopsy was a positive
predictor of interstitial fibrosis at 1 year post-transplant35
E. A new picture of graft damage26-27
a. With developing characterization of subclinical rejection and implications
regarding acute and chronic rejection, there may be more that contributes to
progressive allograft damage than previously speculated
b. Subclinical rejection may represent a “behind the scenes” pathology that
contributes to chronic rejection and acute rejection episodes
F. Treatment29-33
a. In the era of CsA-based maintenance immunosuppression, a single study has
demonstrated benefit with early treatment of subclinical rejection on protocol
biopsy32
i. Prospective, randomized trial of 72 patients demonstrated that patients
who received early protocol biopsies and pulse steroids had significantly
lower serum creatinine and higher GFR at 2 years post-transplant in
comparison to patients who did not receive protocol biopsies
ii. Patients randomized to protocol biopsies also had a lower incidence of
clinical rejection episodes at 2, 3, and 7-12 months post-transplant
iii. Patients in protocol biopsy group also had significantly lower chronic
interstitial and tubular changes at 6 months post-transplant
b. As incidence of subclinical rejection has declined significantly in the era of TAC-
based maintenance immunosuppression regimens, benefit of treating subclinical
rejection has been difficult to demonstrate33
Lyons | { PAGE }
G. Subclinical rejection in the TAC era Study
46 Rush D, Arien D, Boucher A, et al. Lack of benefit of early protocol biopsies in
renal transplant patients receiving TAC and MMF: a randomized study. Am J
Transplant. 2007; 7: 2538-45.
Design Open-label, randomized, prospective, multi-center, parallel-group study in 11
centers in Canada and 1 center in the US
Objective Assess the effect of treating subclinical rejection on protocol biopsy within the first
3 months post-transplant
Interventions Randomized 1:1 to:
Protocol biopsies at 1, 2, 3, and 6 months (Biopsy Arm, BA)
Biopsies at 6 months only (Control Arm, CA)
All patients received TAC, mycophenolate mofetil (MMF), and prednisone for
maintenance immunosuppression
All clinical and subclinical rejection episodes were treated with a 2-week tapering
course of prednisone starting at 200 mg, with the option for investigators to use
methylprednisolone and anti-lymphocyte agents at their discretion
Endpoints Primary: Prevalence of chronic histology at 6 months
Secondary:
Prevalence of subclinical rejection at 6 months
Frequency of biopsy-confirmed or suspected acute rejections within months
0-6
Renal function at 6 months post-transplant
Results ITT Patient Population: 218 patients included (111 in BA, 107 in CA)
Mean age 47.7 years, 67-71% male, 73.8-79.3% Caucasian
No significant (NS) difference between arms with regard to baseline
characteristics
Primary: NS difference in chronic histology scores between both groups (p = 0.09),
although both experienced significant increases in scores between implantation and
month 6
Secondary: Prevalence of SubR at 6 months 9% in the BA vs. 6% in the CA, p =
0.48
Acute rejection rate NS different between arms, with 12 episodes in the BA
and 8 episodes in the CA, respectively (P = 0.44)
NS difference in mean serum creatinine and estimated creatinine clearance
between arms
Author
Conclusions There is no benefit to the procurement of early protocol biopsies in renal
transplant patients receiving TAC, MMF, and prednisone, at least in the
short term, likely due to the low prevalence of SubR
Critique Strengths:
All patients treated with TAC-
based immunosuppression
Multi-center, randomized,
controlled trial
Limitations:
Small sample size
Lack of true control group
Lack of long-term follow-up
Bottom Line In the era of TAC-based immunosuppression, there may be a lower incidence of
subclinical rejection than previously reported in CsA-based regimens. More frequent
protocol biopsies do not prevent subclinical rejection at 6 months. This calls into
question utility of more frequent surveillance biopsies to detect subclinical rejection,
let alone whether or not it should be treated.
Lyons | { PAGE }
CLINICAL QUESTION: IS IT WORTHWHILE TO TREAT SUBCLINICAL
REJECTION?
CLINICAL DATA47-49
Study
47 Kee TYS, Chapman JR, O’Connell PJ et al. Treatment of subclinical rejection
diagnosed by protocol biopsy in kidney transplants. Transplantation. 2006; 82: 36-
42.
Design Retrospective analysis of kidney transplant recipients at a single center between
01/01/2001 and 12/31/2003
Objective Assess the effect of treating subclinical rejection on protocol biopsy within the first
3 months post-transplant
Population Inclusion:
Adult kidney transplant recipient
Combined kidney-pancreas
transplant recipient
Protocol biopsies performed at 1
and 3 months post-transplant
CNI-based immunosuppression
for the first year post-transplant
Exclusion:
Both 1 and 3 month protocol
biopsies not performed
Participation in a clinical drug
trial
Loss to follow-up within 3
months post-transplant
Non-CNI-based maintenance
immunosuppression
Graft loss within first 3 months
post-transplant
Interventions Maintenance immunosuppression
Kidney transplant recipients
o CsA microemulsion 10 mg/kg/day (initial) or TAC 0.2 mg/kg/day
(initial), then adjusted to center goal levels
o MMF 2 gm/day
o Prednisolone 20 mg/day for 3 months, then tapered to 10 mg daily
by 6 months
Kidney-pancreas transplant recipients
o CsA microemulsion 10 mg/kg/day or TAC 0.2 mg/kg/day adjusted
to center goal levels
o MMF 3 gm/day
o Prednisolone 30 mg/day for 3 months, then tapered to 10 mg daily
by 6 months
Treatment of subclinical rejection:
Intravenous methylprednisolone 500 mg daily x 3 days OR increase of
prednisolone dose to 1 mg/kg/day x 5 days (no maximum dose), then
tapered down to baseline dose
OR
Increase in overall immunosuppression (IMS)
o Increase in baseline CsA or TAC dose
o Increase in MMF dose
o Switching CsA to TAC
Endpoints Subclinical rejection at 1 or 3 months post-transplant, defined as histological
evidence of acute rejection or borderline changes in patients with stable renal
function (< 25% change in serum creatinine from baseline)
Lyons | { PAGE }
Subclinical rejection stratified:
Acute (A-SubR) – at least Banff grade 1A
Borderline (B-SubR) – foci of mild tubulitis and at least 10-25%
mononuclear cell interstitial inflammation of the cortex
Results Patient Population:
88 patients included (59 kidney transplant alone, 29 kidney-pancreas)
Mean age 41.3 ± 12.1 years, 57.9% male
45 patients received CsA-based immunosuppression vs. 43 TAC-based
Subclinical Rejection:
46.6% overall incidence of subclinical rejection (n = 41/88)
o A-SubR: 12.5% (n = 11/88)
o B-SubR: 34.1% (n = 30/88)
o Of the 41 episodes of SubR, 33 were identified during the protocol-
defined endpoints, and 8 were identified upon repeat biopsy
25% and 10.2% of SubR was identified at 1 month and 3 months post-
transplant, respectively
SubR occurred in 17 CsA-treated patients and 24 TAC-treated patients
Treatment of Primary Subclinical Rejection Episodes (n = 33)
1 month
(n = 22)
3 months
(n = 9)
12 months
(n = 2)
Increase in IMS alone 4 0
CsA TAC alone 1 2
Pulse steroids alone 2 1
Pulse steroids AND
Increased
IMS
CSATAC
OKT3
10
1
1
1
1
No Treatment 3 5 1
Progression of Subclinical Rejection
Early chronic graft damage was observed in 29.5% (n = 26/88) of all 1-
month protocol biopsies
Higher incidence of chronic graft damage in patients with SubR on 1-month
protocol biopsy (p < 0.005)
Increased IFTA scores in biopsies with SubR vs. no SubR (p < 0.05) at 1
month post-transplant
By 3 month protocol biopsy, inflammation had largely decreased in
comparison to 1 month protocol biopsies
o Significantly lower Banff scores (p < 0.05)
o Chronic allograft damage was increased in the cohort of patients
who were not treated for 1 month SubR compared to those who
were treated (p < 0.05)
Author
Conclusions SubR was common in the study cohort and associated with early chronic
tubulointerstitial damage
Histological progression of injury appeared to be stabilized by treatment
with pulse corticosteroids combined with augmentation of baseline
immunosuppression
Critique Strengths:
Included patients with TAC-
Limitations:
Small sample size
Lyons | { PAGE }
based immunosuppression
Wide variety of methods for
treating subclinical rejection,
allowing for physician freedom
within protocol
Retrospective analysis
Pilot study, observational in
nature
No stratification of results based
on CNI used or organs
transplanted
Wide variety of treatment
options make interpretation and
application of a similar protocol
difficult
No subgroup analysis of risk
factors for subclinical rejection
Bottom Line Treatment of subclinical rejection has impact on the progression of chronic
damage within the renal allograft
Optimal time for protocol biopsy for detection of subclinical rejection is
within 3 months post-transplant
SubR incidence at 1 month post-transplant suggest benefit of protocol
biopsies earlier on in the post-transplant course than 3 months
The optimal treatment regimen for SubR has yet to be elucidated
Study
48 Szederkenyi E, Ivanyi B, Morvay Z, et al. Treatment of subclinical injuries detected
by protocol biopsy improves the long-term kidney allograft function: a single center
prospective randomized clinical trial. Transplantation Proceedings. 2011; 43: 1239-
43.
Design Single center, prospective, randomized trial
Objective Evaluate benefit of early and late protocol biopsies
Evaluate the impact of early treatment of discovered pathologies on allograft
function at 2 and 3 years and long-term (5-year) graft survival
Population Inclusion:
Age > 18 years
Stable graft function at 3 months post-transplant (serum creatinine < 3.39
mg/dL)
No clinical symptoms of rejection within 1 month prior to randomization
Maintenance CNI and MMF-based immunosuppression
Stable immunosuppressive trough levels
o TAC 5-15 ng/mL
o CsA 100-250 ng/mL
Good compliance to treatment
Interventions Patients randomly assigned under controlled allocation 1:1 to two biopsy regimens:
Protocol biopsies at 3 and/or 12 months (biopsy group) within 1 of 3
protocols:
o Protocol biopsy at 3 months only
o Protocol biopsy at 12 months only
o Protocol biopsies at both 3 and 12 months
Clinical surveillance only, no protocol biopsy (control group)
Subclinical rejection: defined as histologic findings consistent with the occurrence
of an acute rejection episode without associated graft dysfunction
Treated with a steroid pulse therapy
Endpoints Outcomes assessed include:
Lyons | { PAGE }
Graft function at 3 months, 1 year, and annually up to 5 years post-
transplant, measured as estimated GFR (eGFR) according to the Cockroft-
Gault method an the Modification of Diet in Renal Disease (MDRD)
formula
Descriptive results of protocol biopsies
Safety of protocol biopsy as defined by incidence of biopsy-related adverse
events
Results Patient Population:
Five-year follow-up data available on 145 patients (N=113 protocol biopsy,
N=51 control)
Majority male (56% and 58% in protocol biopsy and nonbiopsy groups,
respectively), mean age 44 years
NS difference in baseline characteristics, including HLA mismatch,
proportion of patients with PRA ≥ 50%, and induction therapy used
Patients randomized 1:1 to TAC:CsA within groups for maintenance
immunosuppression
o Protocol biopsy group: 59 TAC, 53 CsA
o Control group: 25 TAC, 24 CsA
Graft Function:
NS difference in eGFR at 1 and 2 years post-transplant between groups
Significantly lower serum creatinine (1.80 ± 0.51 mg/dL in protocol biopsy
group vs. 2.45 ± 1.05 mg/dL in control group, p=0.003) and greater eGFR
observed in protocol biopsy arm (46.0 ± 13.8 mL/min/1.73 m2 in protocol
biopsy group vs. 35.4 ± 15.0 mL/min/1.73 m2 in control group, p = 0.002) at
3 years post-transplant
Subclinical Rejection
65 cases of subclinical acute rejection identified by 1 year post-transplant all
treated with pulse steroids
Numerically higher amount of SubR identified at 12 months post-transplant,
but not statistically significant (p = 0.08)
Safety:
7 patients experienced biopsy-related complications
o All patients were discharged after the 4-hour post-biopsy
observation time
Author
Conclusions Protocol biopsy is an excellent method for the early diagnosis of disorders in
the transplanted kidney and to monitor the effects of immunosuppression
The protocol biopsy, followed by appropriate treatment, promotes
preservation of kidney allograft function and therefore improves long-term
graft survival
Critique Strengths:
Prospective, randomized study
All subclinical rejection
episodes treated the same, with
pulse steroid therapy
Long-term follow-up
Inclusion and randomization of
patients to TAC and CsA
Limitations:
Single center, small sample size
No definition of graft
dysfunction
Lack of pre-defined outcomes
No stratification based on CNI
No stratification of results based
on protocol biopsy regimen used
“Pulse steroids” is ambiguous
Bottom Line Use of protocol biopsies within the first year post-transplant, as well as
Lyons | { PAGE }
prompt treatment of subclinical rejection has beneficial effect on long-term
(> 2 years) renal graft function
o There appears to be no beneficial effect on allograft function at 1
year post-transplant
Study
49 Gigliotti P, Lofaro D, Leone F, et al. Early subclinical rejection treated with low
dose i.v. steroids is not associated to graft survival impairment: 13 years’ experience
at a single center. J Nephrol. 2016; 29: 443-9.
Design Single center, retrospective study
Objective Evaluate the incidence of early subclinical rejection revealed by protocol biopsy at 1
months post kidney transplantation, factors potentially involved in the manifestation
of SubR, and evaluate the association between SubR previously treated with low-
dose intravenous steroids and long-term graft survival
Population Inclusion:
Primary kidney transplant
Deceased donor
Protocol biopsy at day 30 post-
transplant
Exclusion:
Primary non function
For-cause biopsy before day 30
post-transplant
Interventions SubR defined as absence of functional deterioration and with presence of histologic
findings indicative of rejection on the basis of tubulitis (t) and mononuclear cell
infiltration (i) scores:
t score ≥ 1 and i score > 0 in the absence of functional deterioration (< 15%
decrease in serum creatinine) classified as SubR
Immunosuppression regimens:
Induction: basiliximab
Maintenance:
o CNI (TAC or CsA) adjusted to levels:
TAC trough levels of 5-15 ng/mL
CsA C0 levels of 150-300 ng/mL
CsA C2 levels of 1400-1800 ng/mL early post-transplant
and 800-1200 ng/mL later after transplant
o MMF 1.5 gm/day
o Steroids
Treatment:
SubR: methylprednisolone 250 mg/day intravenous for 3 days
Acute rejection: methylprednisolone 500 mg/day intravenous for 3 days
o Anti-thymocyte globulin was used as clinically and histologically
indicated
Patients divided into normal, SubR, and acute rejection groups according to Banff
classification on protocol biopsy for analysis
Endpoints 10-year graft survival
Cox-proportional hazards regression model to determine factors associated
with graft survival
Results Patient population:
Italian
174 patients screened, 159 patients underwent protocol biopsy
59.8% male, mean age 46.15 ± 12.2 years
69 patients (39.7%) experienced DGF requiring ≥ 1 days on dialysis
113 of 174 patients received TAC as CNI (64.9%)
Lyons | { PAGE }
Outcomes of interest:
Protocol biopsy results (n =159)
o Normal histology: 142 patients (89.3%)
o SubR: 17 patients (10.7%), all treated with pulse steroids per
protocol
10 patients with functional changes
o 2 with CsA toxicity
o 8 with acute rejection
NS difference in SubR between patients treated with TAC vs. CsA (9.8%
treated with TAC vs. 9.7% treated with CsA, p = 0.202)
Factors associated with SubR (multivariate analysis):
o Donor age: OR 1.04 (95% CI 1.01-1.09)
o DGF: OR 1.08 (95% CI 1.03-1.12)
10-year graft survival:
o NS difference between SubR group and normal histology group
o Acute rejection group had a significantly lower 10-year graft
survival in comparison to both SubR and normal histology groups
o Factors associated with 10-year graft failure (multivariate analysis):
Donor age: HR 1.03 (95% CI 1.01-1.05)
DGF: HR 1.57 (95% CI 1.04-2.22)
Acute rejection: HR 5.22 (95% CI 1.70-16.01)
Subclinical rejection WITH TREATMENT was not
independently associated with 10-year graft failure
Author
Conclusions
An early protocol biopsy performed 1 month after renal transplantation is a useful
tool to detect subclinical rejection, and anti-rejection treatment for SubR with low-
dose i.v. pulse steroids could be an appropriate strategy to improve kidney transplant
graft survival in the long term
Critique Strengths:
All patients with SubR received
identical treatment
Greater proportion of patient
population received TAC,
mimicking current clinical
practice
Long-term follow up
Limitations:
No evaluation of graft function
outside of overall graft survival
Single-center, Italian patient
population hinders external
validity
Retrospective
No true control group
Bottom Line Results support the practice of using early (< 3 month post-transplant)
protocol biopsies to evaluate early subclinical changes in renal transplant
recipients
Standardized treatment of subclinical rejection with low-dose pulse steroids
and lack of significant difference in graft survival at 10-years post-transplant
suggests that pre-emptive treatment is able to attenuate or even reverse these
subclinical changes
Patients with treated subclinical rejection behave more like patients without
rejection
A prospective, randomized trial would be the gold-standard to assess this
hypothesis
Lyons | { PAGE }
SUMMARY
Incidence of SubR was drastically reduced with the introduction of TAC and MMF-based
maintenance immunosuppression regimens
Few well-designed studies have directly evaluated the benefit of treatment of SubR
The optimal time for detection of SubR is within the first 3 months post-transplant
Early treatment of SubR has negligible benefit on renal function at 1 year post-transplant,
but is associated with significant benefit on long-term (> 2 year) renal function
Pulse steroids are able to attenuate and reverse SubR episodes
RECOMMENDATION
Patients at high immunologic risk for the development of CAN/IFTA should be
monitored with protocol biopsies post-transplant
o Positive crossmatch
o ABO-incompatible
o Delayed graft function
For centers utilizing protocol biopsies after renal transplant, biopsies should be performed
within 3 months post-transplant
For centers performing routine protocol biopsies within the first year post-transplant,
SubR should be treated if detected
o Methylprednisolone 250-500 mg daily for 3 days (similar to treatment for acute
rejection)
o Optimization of immunosuppression
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Appendix A. Abbreviations
ACR: Acute cellular rejection
AMR: Antibody-mediated rejection
APC: Antigen-presenting cell
A-SubR: Acute subclinical rejection
B-SubR: Borderline subclinical rejection
CAN: Chronic allograft nephropathy
CNI: Calcineurin inhibitor
CsA: Cyclosporine
DGF: Delayed graft function
DSA: Donor specific antibody
eGFR: Estimated glomerular filtration rate
GFR: Glomerular filtration rate
HLA: Human leukocyte antigen
IFTA: Interstitial fibrosis and tubular atrophy
IL-2RA: Interleukin 2 receptor antagonist
IVIG: Intravenous immune globulin
MAC: Membrane attack complex
MMF: Mycophenolate mofetil
SubR: Subclinical rejection
TAC: Tacrolimus
Appendix B. Banff ’97 Classification of Renal Allograft Pathology, 2009 Update9
Diagnosis Histologic Characteristics and Grading
Normal Normal histology of biopsy sample
Borderline Changes
“Suspicious” for acute t-cell mediated
rejection:
1. No intimal arteritis
2. Foci of tubulitis with minor interstitial
infiltration
Interstitial infiltration with mild tubulitis
Acute T-cell-mediated rejection
IA. Interstitial inflammation (> 25% of
parenchyma) and foci of moderate tubulitis
IB. Interstitial inflammation (>25% of
parenchyma) and foci of severe tubulitis
IIA. Mild to moderate intimal arteritis
IIB. Severe intimal arteritis comprising >25%
of luminal area
III. Transmural arteritis and/or arterial fibrinoid
change and necrosis of medial smooth muscle
cells with accompanying lymphocytic
inflammation