- Email: [email protected]
C4d Deposits in Borderline Rejection: An Early Marker for Chronic Renal Dysfunction?
C4d Deposits in Borderline Rejection: An Early Marker for Chronic Renal Dysfunction? W.L.V. Sampaio and M. Mazzali* Division of Nephrology, Department of Medicine, School of Medical Sciences, State University of Campinas, Campinas, São Paolo, Brazil
ABSTRACT The impact of borderline rejection in renal graft remains controversial. The aim of this study was to analyze the presence of C4d deposits in peritubular capillaries and macrophage infiltration in renal biopsies with diagnosis of borderline rejection ant its effect on graft function. Thirty-one renal transplant recipients with a diagnosis of borderline rejection were included. Initial and sequential biopsies were analyzed for morphology, C4d, and macrophage staining and compared with clinical data. Initial biopsies showed 12 samples to be C4d positive, associated with a higher incidence of delayed graft function, earlier posttransplantation time, higher acute tubular necrosis score, capillaritis, and glomerular macrophage infiltration, and a lower level of tubulitis, interstitial fibrosis, and tubular atrophy compared with the C4d-negative samples. In sequential biopsies, 5 patients from the negative group turned C4d positive. Patients with 1 positive C4d biopsy (n ¼ 17) showed lower renal graft function at 6 months (1.8 0.8 vs 1.4 0.5 mg/dL; P < .01), 1 year (2.1 1 vs 1.5 0.5 mg/dL; P < .01), and 2 years (2.3 1.3 vs 1.5 0.7 mg/dL; P < .05) of follow-up. The expression of C4d in peritubular capillaries of renal biopsies classified as borderline rejection was associated with a worse prognosis for the renal allograft.
A
CUTE borderline rejection is defined as the presence of interstitial inflammation in 10%e25% of renal parenchyma, focal tubulitis with <4 infiltrating cells/tubular section, and absence of intimal arteritis [1]. Natural history and the impact of borderline rejection in long-term graft function remain unclear [2]. Although some studies showed similar 2-year graft function in patients with borderline rejection compared with biopsies with normal renal histology, others showed that renal function was similar to acute rejection. Also, sequential biopsies showed a progression from borderline to acute cellular rejection [2e4]. These results vary according to study population (protocol vs indication biopsy), time for post-transplantation biopsy, previous treatment of rejection, presence of acute tubular necrosis (ATN), and/or signs of drug toxicity [3,4]. Therapy for borderline rejection also is controversial. Although isolated borderline changes remain untreated, in the presence of graft dysfunction therapy is common sense because of the risk of progress to rejection [5]. Recently, presence of C4d deposits in peritubular capillaries (PTCs) is associated with worse graft function. These deposits are indirect signs of antibody-mediated complement activation
and persist in tissue even after the disappearance of the antibody [6e8]. In biopsies with borderline changes, and graft dysfunction, C4d is observed in 18%-46% [4] and can be associated with glomerulitis and glomerular macrophage infiltration, with a higher risk for chronic rejection and graft loss [9e13]. In the present study, we hypothesized that the presence of C4d deposits in PTCs and macrophage infiltration in kidney allograft biopsies, classified as borderline, were associated with reduced graft function and survival. MATERIAL AND METHODS Renal graft biopsies with diagnosis of borderline changes were selected from the Renal Transplant Unit Database. The protocol was approved by the local Ethics Committee.
*Address correspondence to Marilda Mazzali, MD, Division of Nephrology, Department of Medicine, School of Medical Sciences, Rua Tessália Vieira de Camargo, 126, State University of Campinas, CEP 13083-970, Campinas, SP, Brazil. E-mail: [email protected]
0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2014.05.014
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1710
Transplantation Proceedings, 46, 1710e1712 (2014)
C4d DEPOSITS IN BORDERLINE REJECTION Inclusion criteria were: biopsy indicated by graft dysfunction within 6 months after transplantation; absence of earlier biopsyproven acute rejection or acute rejection therapy. Exclusion criteria were: pediatric recipients (<18 years old), earlier acute rejection, and inadequate material for histologic analysis. Thirtyone patients were included. Sequential biopsies from these patients were also analyzed.
Morphologic Analysis Biopsies were reclassified according to Banff 1997 and 2005 criteria and with the use of a semiquantitative score for tubular/interstitial lesions. Biopsy specimens were recovered from paraffin blocks and stained with C4d and macrophage antibodies, following a local staining protocol. Briefly, slides were incubated overnight with primary antibody for C4d (polyclonal rabbit antiehuman C4d IgG, 1:1,000; Biomedica Gruppe, Austria) or macrophages (monoclonal mouse antiehuman CD68 IgG, 1:100; Novocastra, United Kingdom), followed by specific secondary antibody incubation for 30 minutes. Diaminobenzidine was used as a chromogen, and slides were counterstained with Harris hematoxylin. Staining was quantified with the use of computer-assisted analysis (KS 300; Zeiss). C4d was classified according to staining in the perimeter of PTCs as absent, focal (<50%), or diffuse (>50%), excluding areas of interstitial fibrosis. Macrophages were quantified by counting the number of macrophages in the interstitium, corrected for 100 cortical tubules. Values were expressed as cells/mm2. Glomerular macrophages were quantified as the absolute number of macrophages/total glomeruli analyzed. Clinical data were collected from medical records and included information about the renal transplantation, donor and patient demographics, initial immunosuppressive therapy, occurrence of delayed graft function, need for dialysis, and immunosuppressive therapy.
Statistical Analysis Results were expressed as mean standard deviation. Data were analyzed with the use of Student t test, chi-square, and Fisher exact test when indicated, with the use of the Stat View 2.0 program. Statistical significance was considered to be P < .05.
RESULTS
Patients were mostly male (n ¼ 23; 74%), recipients of first renal transplant (n ¼ 29; 93%) from a deceased donor (n ¼ 24; 77%), with a mean age of 37 11 years old (range, 20e56). Only 1 patient had anti-HLA antibodies detectable, which were not donor specific. Of the 31 initial renal biopsies classified as borderline, 12 (39%) showed positive C4d staining in PTCs, and 19 (61%) were C4d negative. There were no significant differences in demographics from patients from C4d-positive or -negative groups. However, we observed a higher incidence of delayed graft function in the C4dþ group (92% vs 74%; P < .05), which may explain the early indication of biopsy in this group (10 3.1 vs 28 40 days; P < .05; Table 1). Morphologic analysis of periodic acideSchiff and hematoxylin-eosin stained slides showed a higher incidence of degenerative ATN and capilaritis in the C4dþ group, whereas the C4d group had a higher score for interstitial inflammation. [Table 1]. Analysis of sequential biopsies
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showed a lower level of interstitial fibrosis and tubular atrophy in the C4dþ group. Other morphologic parameters were similar between groups. Interstitial infiltration of macrophages was similar in C4dþ and C4d groups. However, glomerular macrophage infiltration was higher in the C4dþ group (42% vs 25%; P < .05). We also analyzed the impact of the C4d deposits on longterm graft function and survival. Patients with 1 biopsy with C4d in PTCs, in early or sequential biopsies were considered to be C4dþ. During follow-up, 12 patients persisted C4d, whereas 7 turned C4dþ. Renal function, measured by serum creatinine and estimated glomerular filtration rate (eGFR; Modification of Diet in Renal Disease formula) showed reduced graft function in the C4dþ group at 6, 12, and 24 months of follow-up (P < .05). After a 2-year period, 4 grafts were lost due to chronic dysfunction, with a higher incidence in the C4d þ group (16% vs 8%; P ¼ ns). DISCUSSION
Diagnosis of borderline changes in indication allograft biopsies raises a question of whether these findings resemble an inadequate sample or are early markers of acute rejection. In this study, we analyzed the presence of C4d deposits and macrophage infiltration in allograft biopsies classified as borderline. Morphologic analysis showed a lower incidence of interstitial inflammation in C4dþ. C4d deposits in PTCs without interstitial inflammation were previously described and can be an indicator of antibody-mediated injury independent from cell activation and infiltration [6,9]. In the present study, early biopsies showed interstitial inflammation without tubulitis. Sequential biopsies in the C4d group showed mild or moderate tubulitis (Banff 1A or 1B
Table 1. Demographics of Renal Transplant Recipients and Histologic Parameters, According to C4d Deposition in Renal Biopsies
Recipient age (y) Sex (male:female) Blood transfusion >5 units [n (%)] Donor source (living:deceased) Donor age (y) Cold ischemia time (h) Delayed graft function [n (%)] Time for first allograft biopsy (d) No of glomeruli/biopsy No of arteries/biopsy Interstitial inflammation (%) Degenerative ATN (score) Regenerative ATN (score) Interstitial fibrosis (score) Tubular atrophy (score) Peritubular capilaritis (%) Abbreviation: ATN, acute tubular necrosis. *P < .05.
C4d Negative (n ¼ 19)
C4d Positive (n ¼ 12)
40 10 14:5 6 (32%) 5:14 31 11 21 6.7 14 (74%) 28 40 8.2 6.0 1.7 1.2 8.6 7.6 0.3 0.7 1.0 0.8 1.4 0.8 0.4 0.7 21%
32 8.7 9:3 3 (25%) 2:10 35 13 20 6.7 11 (92%)* 10 3.1* 7.8 5.1 1.0 1.0 4.7 3.6* 1.4 1.4* 1.3 1.0 1.7 0.8 1.0 1.0 41%*
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acute rejection), reinforcing the idea that borderline changes without C4d could be an early presentation of cellmediated acute rejection. The higher incidence of delayed graft function and degenerative ATN in the C4dþ group is compatible with the early indication of biopsy in this group, usually 14 days before the biopsies in the C4d group. ATN can be an initial stimulus for acute rejection, by exposing antigenpresenting cells in areas of tubular cell necrosis. Local inflammatory response, mediated by dendritic cells, T cells, and macrophages, results in cell attack, characterized by tubulitis or vascular afflux of T cells. However, in cases of antibody mediated acute rejection, complement activation and local tissue response can be limited to PTCs, with the presence of minimal histologic changes but increased capillaritis and C4d deposition [8]. In the present series, incidence of capillaritis was significantly higher in C4dþ group. The persistence of ATN was associated with a need for sequential biopsies in 9 patients from the C4dþ group, with a diagnosis of ATN associated with borderline findings in 5 cases. In all cases, we observed the persistence of C4d deposits in PTCs. Interestingly, in the C4d group, sequential biopsies, performed in 6 patients, were diagnosed as acute cellular rejection or borderline changes. C4d was observed in 3 cases, with a higher frequency in borderline rejection. The biggest problem with the diagnosis of borderline changes in sequential biopsies was the presence of areas of tubular atrophy and interstitial fibrosis which could be a confounding factor for interstitial infiltrates. Interstitial scarring is considered to be nonspecific and can be a consequence of ischemic injury, hypertension, or calcineurin inhibitor toxicity [14]. Borderline changes are a common finding in protocol biopsies and considered to be subclinical rejection with a negative impact on long-term graft function. In the present series with indication biopsies, borderline changes were not classified as subclinical rejection. However, the presence of C4dþ in PTCs could be considered to be an early marker for antibody-mediated rejection, increasing the risk for chronic rejection. Considering the hypothesis that C4d deposits could be an early marker for antibody-mediated rejection, we analyzed the macrophage infiltration in these biopsies. Earlier studies suggested that glomerular macrophage infiltration was associated with antibody-mediated glomerulitis and increased risk for chronic allograft rejection or glomerulonephritis [12,13]. In the present series, glomerular macrophage infiltration was significantly higher in the C4dþ group, despite the similar interstitial infiltration. To analyze the impact of C4d deposits in long-term graft survival, we measured renal function by serum creatinine or eGFR. We observed that patients from the C4dþ group had reduced renal function at 6, 12, and 24 months, with a
SAMPAIO AND MAZZALI
higher incidence of graft loss due to chronic rejection, suggesting that C4d deposits in PTCs can predict the outcome independently from histologic changes [15]. Limitations of this study are the retrospective design with a small sample and the lack of donor-specific antibody quantification to confirm the diagnosis of antibodymediated rejection with borderline changes. However, the data provide evidence that C4d deposits in borderline changes in the presence of graft dysfunction are associated with unfavorable renal function and a higher risk of chronic rejection and graft loss.
REFERENCES [1] Solez K, Axelsen RA, Benediktsson H, et al. International standardization of criteria for the histologic diagnosis of renal allograft rejection: the Banff working classification of kidney transplant pathology. Kidney Int 1993;44:411e22. [2] Saad R, Gritsch HA, Shapiro R, et al. Clinical significance of renal allograft biopsies with “borderline changes” as defined in the Banff schema. Transplantation 1997;64:992e5. [3] Meehan SM, Siegel CT, Aronson AJ, et al. The relationship of untreated borderline infiltrates by Banff criteria to acute rejection in renal allograft biopsies. J Am Soc Nephrol 1999;10:1806e14. [4] Mengel M, Bogers J, Bosmans JL, et al. ESPRIT Group. Incidence of C4d stain in protocol biopsies from renal allografts: results from a multicenter trial. Am J Transplant 2005;5:1050e6. [5] Beimler J, Zeier M. Borderline rejection after renal transplantationdto treat or not to treat. Clin Transplant 2009;23(Suppl 21):19e25. [6] Feucht HE, Felber E, Gokel MJ, et al. Vascular deposition of complement-split products in kidney allografts with cell-mediated rejection. Clin Exp Immunol 1991;86:464e70. [7] Regele H, Exner M, Watschinger B, et al. Endothelial C4d deposition is associated with inferior kidney allograft outcome independently of cellular rejection. Nephrol Dial Transplant 2001;16:2058e66. [8] Mauiyyedi S, Crespo M, Collins AB, et al. Acute humoral rejection in kidney transplantation: II. Morphology, immunopathology and pathologic classification. J Am Soc Nephrol 2002;13:779e87. [9] Herzenberg AM, Gill JS, Djurdjev O, Magil AB. C4d deposition in acute rejection: an independent long-term prognostic factor. J Am Soc Nephrol 2002;13:234e41. [10] Lorenz M, Regele H, Schillinger M, et al. Risk factors for capillary C4d deposition in kidney allografts: evaluation of a large study cohort. Transplantation 2004;78:447e52. [11] Nickeleit V, Zeiler M, Gudat F, et al. Detection of the complement degradation product C4d in renal allografts: diagnostic and therapeutic implications. J Am Soc Nephrol 2002;13:242e51. [12] Magil AB, Tinckam K. Monocytes and peritubular capillary C4d deposition in acute renal allograft rejection. Kidney Int 2003;63:1888e93. [13] Tinckam KJ, Djurdjev O, Magil AB. Glomerular monocytes predict worse outcomes after acute renal allograft rejection independent of C4d status. Kidney Int 2005;68:1866e74. [14] Solez K, Colvin RB, Racusen LC, et al. Banff ’05 Meeting Report: differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy (“CAN”). Am J Transplant 2007;7:518e26. [15] Haas M. The significance of C4d staining with minimal histologic abnormalities. Curr Opin Organ Transplant 2010;15:21e7.
ABSTRACT The impact of borderline rejection in renal graft remains controversial. The aim of this study was to analyze the presence of C4d deposits in peritubular capillaries and macrophage infiltration in renal biopsies with diagnosis of borderline rejection ant its effect on graft function. Thirty-one renal transplant recipients with a diagnosis of borderline rejection were included. Initial and sequential biopsies were analyzed for morphology, C4d, and macrophage staining and compared with clinical data. Initial biopsies showed 12 samples to be C4d positive, associated with a higher incidence of delayed graft function, earlier posttransplantation time, higher acute tubular necrosis score, capillaritis, and glomerular macrophage infiltration, and a lower level of tubulitis, interstitial fibrosis, and tubular atrophy compared with the C4d-negative samples. In sequential biopsies, 5 patients from the negative group turned C4d positive. Patients with 1 positive C4d biopsy (n ¼ 17) showed lower renal graft function at 6 months (1.8 0.8 vs 1.4 0.5 mg/dL; P < .01), 1 year (2.1 1 vs 1.5 0.5 mg/dL; P < .01), and 2 years (2.3 1.3 vs 1.5 0.7 mg/dL; P < .05) of follow-up. The expression of C4d in peritubular capillaries of renal biopsies classified as borderline rejection was associated with a worse prognosis for the renal allograft.
A
CUTE borderline rejection is defined as the presence of interstitial inflammation in 10%e25% of renal parenchyma, focal tubulitis with <4 infiltrating cells/tubular section, and absence of intimal arteritis [1]. Natural history and the impact of borderline rejection in long-term graft function remain unclear [2]. Although some studies showed similar 2-year graft function in patients with borderline rejection compared with biopsies with normal renal histology, others showed that renal function was similar to acute rejection. Also, sequential biopsies showed a progression from borderline to acute cellular rejection [2e4]. These results vary according to study population (protocol vs indication biopsy), time for post-transplantation biopsy, previous treatment of rejection, presence of acute tubular necrosis (ATN), and/or signs of drug toxicity [3,4]. Therapy for borderline rejection also is controversial. Although isolated borderline changes remain untreated, in the presence of graft dysfunction therapy is common sense because of the risk of progress to rejection [5]. Recently, presence of C4d deposits in peritubular capillaries (PTCs) is associated with worse graft function. These deposits are indirect signs of antibody-mediated complement activation
and persist in tissue even after the disappearance of the antibody [6e8]. In biopsies with borderline changes, and graft dysfunction, C4d is observed in 18%-46% [4] and can be associated with glomerulitis and glomerular macrophage infiltration, with a higher risk for chronic rejection and graft loss [9e13]. In the present study, we hypothesized that the presence of C4d deposits in PTCs and macrophage infiltration in kidney allograft biopsies, classified as borderline, were associated with reduced graft function and survival. MATERIAL AND METHODS Renal graft biopsies with diagnosis of borderline changes were selected from the Renal Transplant Unit Database. The protocol was approved by the local Ethics Committee.
*Address correspondence to Marilda Mazzali, MD, Division of Nephrology, Department of Medicine, School of Medical Sciences, Rua Tessália Vieira de Camargo, 126, State University of Campinas, CEP 13083-970, Campinas, SP, Brazil. E-mail: [email protected]
0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2014.05.014
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
1710
Transplantation Proceedings, 46, 1710e1712 (2014)
C4d DEPOSITS IN BORDERLINE REJECTION Inclusion criteria were: biopsy indicated by graft dysfunction within 6 months after transplantation; absence of earlier biopsyproven acute rejection or acute rejection therapy. Exclusion criteria were: pediatric recipients (<18 years old), earlier acute rejection, and inadequate material for histologic analysis. Thirtyone patients were included. Sequential biopsies from these patients were also analyzed.
Morphologic Analysis Biopsies were reclassified according to Banff 1997 and 2005 criteria and with the use of a semiquantitative score for tubular/interstitial lesions. Biopsy specimens were recovered from paraffin blocks and stained with C4d and macrophage antibodies, following a local staining protocol. Briefly, slides were incubated overnight with primary antibody for C4d (polyclonal rabbit antiehuman C4d IgG, 1:1,000; Biomedica Gruppe, Austria) or macrophages (monoclonal mouse antiehuman CD68 IgG, 1:100; Novocastra, United Kingdom), followed by specific secondary antibody incubation for 30 minutes. Diaminobenzidine was used as a chromogen, and slides were counterstained with Harris hematoxylin. Staining was quantified with the use of computer-assisted analysis (KS 300; Zeiss). C4d was classified according to staining in the perimeter of PTCs as absent, focal (<50%), or diffuse (>50%), excluding areas of interstitial fibrosis. Macrophages were quantified by counting the number of macrophages in the interstitium, corrected for 100 cortical tubules. Values were expressed as cells/mm2. Glomerular macrophages were quantified as the absolute number of macrophages/total glomeruli analyzed. Clinical data were collected from medical records and included information about the renal transplantation, donor and patient demographics, initial immunosuppressive therapy, occurrence of delayed graft function, need for dialysis, and immunosuppressive therapy.
Statistical Analysis Results were expressed as mean standard deviation. Data were analyzed with the use of Student t test, chi-square, and Fisher exact test when indicated, with the use of the Stat View 2.0 program. Statistical significance was considered to be P < .05.
RESULTS
Patients were mostly male (n ¼ 23; 74%), recipients of first renal transplant (n ¼ 29; 93%) from a deceased donor (n ¼ 24; 77%), with a mean age of 37 11 years old (range, 20e56). Only 1 patient had anti-HLA antibodies detectable, which were not donor specific. Of the 31 initial renal biopsies classified as borderline, 12 (39%) showed positive C4d staining in PTCs, and 19 (61%) were C4d negative. There were no significant differences in demographics from patients from C4d-positive or -negative groups. However, we observed a higher incidence of delayed graft function in the C4dþ group (92% vs 74%; P < .05), which may explain the early indication of biopsy in this group (10 3.1 vs 28 40 days; P < .05; Table 1). Morphologic analysis of periodic acideSchiff and hematoxylin-eosin stained slides showed a higher incidence of degenerative ATN and capilaritis in the C4dþ group, whereas the C4d group had a higher score for interstitial inflammation. [Table 1]. Analysis of sequential biopsies
1711
showed a lower level of interstitial fibrosis and tubular atrophy in the C4dþ group. Other morphologic parameters were similar between groups. Interstitial infiltration of macrophages was similar in C4dþ and C4d groups. However, glomerular macrophage infiltration was higher in the C4dþ group (42% vs 25%; P < .05). We also analyzed the impact of the C4d deposits on longterm graft function and survival. Patients with 1 biopsy with C4d in PTCs, in early or sequential biopsies were considered to be C4dþ. During follow-up, 12 patients persisted C4d, whereas 7 turned C4dþ. Renal function, measured by serum creatinine and estimated glomerular filtration rate (eGFR; Modification of Diet in Renal Disease formula) showed reduced graft function in the C4dþ group at 6, 12, and 24 months of follow-up (P < .05). After a 2-year period, 4 grafts were lost due to chronic dysfunction, with a higher incidence in the C4d þ group (16% vs 8%; P ¼ ns). DISCUSSION
Diagnosis of borderline changes in indication allograft biopsies raises a question of whether these findings resemble an inadequate sample or are early markers of acute rejection. In this study, we analyzed the presence of C4d deposits and macrophage infiltration in allograft biopsies classified as borderline. Morphologic analysis showed a lower incidence of interstitial inflammation in C4dþ. C4d deposits in PTCs without interstitial inflammation were previously described and can be an indicator of antibody-mediated injury independent from cell activation and infiltration [6,9]. In the present study, early biopsies showed interstitial inflammation without tubulitis. Sequential biopsies in the C4d group showed mild or moderate tubulitis (Banff 1A or 1B
Table 1. Demographics of Renal Transplant Recipients and Histologic Parameters, According to C4d Deposition in Renal Biopsies
Recipient age (y) Sex (male:female) Blood transfusion >5 units [n (%)] Donor source (living:deceased) Donor age (y) Cold ischemia time (h) Delayed graft function [n (%)] Time for first allograft biopsy (d) No of glomeruli/biopsy No of arteries/biopsy Interstitial inflammation (%) Degenerative ATN (score) Regenerative ATN (score) Interstitial fibrosis (score) Tubular atrophy (score) Peritubular capilaritis (%) Abbreviation: ATN, acute tubular necrosis. *P < .05.
C4d Negative (n ¼ 19)
C4d Positive (n ¼ 12)
40 10 14:5 6 (32%) 5:14 31 11 21 6.7 14 (74%) 28 40 8.2 6.0 1.7 1.2 8.6 7.6 0.3 0.7 1.0 0.8 1.4 0.8 0.4 0.7 21%
32 8.7 9:3 3 (25%) 2:10 35 13 20 6.7 11 (92%)* 10 3.1* 7.8 5.1 1.0 1.0 4.7 3.6* 1.4 1.4* 1.3 1.0 1.7 0.8 1.0 1.0 41%*
1712
acute rejection), reinforcing the idea that borderline changes without C4d could be an early presentation of cellmediated acute rejection. The higher incidence of delayed graft function and degenerative ATN in the C4dþ group is compatible with the early indication of biopsy in this group, usually 14 days before the biopsies in the C4d group. ATN can be an initial stimulus for acute rejection, by exposing antigenpresenting cells in areas of tubular cell necrosis. Local inflammatory response, mediated by dendritic cells, T cells, and macrophages, results in cell attack, characterized by tubulitis or vascular afflux of T cells. However, in cases of antibody mediated acute rejection, complement activation and local tissue response can be limited to PTCs, with the presence of minimal histologic changes but increased capillaritis and C4d deposition [8]. In the present series, incidence of capillaritis was significantly higher in C4dþ group. The persistence of ATN was associated with a need for sequential biopsies in 9 patients from the C4dþ group, with a diagnosis of ATN associated with borderline findings in 5 cases. In all cases, we observed the persistence of C4d deposits in PTCs. Interestingly, in the C4d group, sequential biopsies, performed in 6 patients, were diagnosed as acute cellular rejection or borderline changes. C4d was observed in 3 cases, with a higher frequency in borderline rejection. The biggest problem with the diagnosis of borderline changes in sequential biopsies was the presence of areas of tubular atrophy and interstitial fibrosis which could be a confounding factor for interstitial infiltrates. Interstitial scarring is considered to be nonspecific and can be a consequence of ischemic injury, hypertension, or calcineurin inhibitor toxicity [14]. Borderline changes are a common finding in protocol biopsies and considered to be subclinical rejection with a negative impact on long-term graft function. In the present series with indication biopsies, borderline changes were not classified as subclinical rejection. However, the presence of C4dþ in PTCs could be considered to be an early marker for antibody-mediated rejection, increasing the risk for chronic rejection. Considering the hypothesis that C4d deposits could be an early marker for antibody-mediated rejection, we analyzed the macrophage infiltration in these biopsies. Earlier studies suggested that glomerular macrophage infiltration was associated with antibody-mediated glomerulitis and increased risk for chronic allograft rejection or glomerulonephritis [12,13]. In the present series, glomerular macrophage infiltration was significantly higher in the C4dþ group, despite the similar interstitial infiltration. To analyze the impact of C4d deposits in long-term graft survival, we measured renal function by serum creatinine or eGFR. We observed that patients from the C4dþ group had reduced renal function at 6, 12, and 24 months, with a
SAMPAIO AND MAZZALI
higher incidence of graft loss due to chronic rejection, suggesting that C4d deposits in PTCs can predict the outcome independently from histologic changes [15]. Limitations of this study are the retrospective design with a small sample and the lack of donor-specific antibody quantification to confirm the diagnosis of antibodymediated rejection with borderline changes. However, the data provide evidence that C4d deposits in borderline changes in the presence of graft dysfunction are associated with unfavorable renal function and a higher risk of chronic rejection and graft loss.
REFERENCES [1] Solez K, Axelsen RA, Benediktsson H, et al. International standardization of criteria for the histologic diagnosis of renal allograft rejection: the Banff working classification of kidney transplant pathology. Kidney Int 1993;44:411e22. [2] Saad R, Gritsch HA, Shapiro R, et al. Clinical significance of renal allograft biopsies with “borderline changes” as defined in the Banff schema. Transplantation 1997;64:992e5. [3] Meehan SM, Siegel CT, Aronson AJ, et al. The relationship of untreated borderline infiltrates by Banff criteria to acute rejection in renal allograft biopsies. J Am Soc Nephrol 1999;10:1806e14. [4] Mengel M, Bogers J, Bosmans JL, et al. ESPRIT Group. Incidence of C4d stain in protocol biopsies from renal allografts: results from a multicenter trial. Am J Transplant 2005;5:1050e6. [5] Beimler J, Zeier M. Borderline rejection after renal transplantationdto treat or not to treat. Clin Transplant 2009;23(Suppl 21):19e25. [6] Feucht HE, Felber E, Gokel MJ, et al. Vascular deposition of complement-split products in kidney allografts with cell-mediated rejection. Clin Exp Immunol 1991;86:464e70. [7] Regele H, Exner M, Watschinger B, et al. Endothelial C4d deposition is associated with inferior kidney allograft outcome independently of cellular rejection. Nephrol Dial Transplant 2001;16:2058e66. [8] Mauiyyedi S, Crespo M, Collins AB, et al. Acute humoral rejection in kidney transplantation: II. Morphology, immunopathology and pathologic classification. J Am Soc Nephrol 2002;13:779e87. [9] Herzenberg AM, Gill JS, Djurdjev O, Magil AB. C4d deposition in acute rejection: an independent long-term prognostic factor. J Am Soc Nephrol 2002;13:234e41. [10] Lorenz M, Regele H, Schillinger M, et al. Risk factors for capillary C4d deposition in kidney allografts: evaluation of a large study cohort. Transplantation 2004;78:447e52. [11] Nickeleit V, Zeiler M, Gudat F, et al. Detection of the complement degradation product C4d in renal allografts: diagnostic and therapeutic implications. J Am Soc Nephrol 2002;13:242e51. [12] Magil AB, Tinckam K. Monocytes and peritubular capillary C4d deposition in acute renal allograft rejection. Kidney Int 2003;63:1888e93. [13] Tinckam KJ, Djurdjev O, Magil AB. Glomerular monocytes predict worse outcomes after acute renal allograft rejection independent of C4d status. Kidney Int 2005;68:1866e74. [14] Solez K, Colvin RB, Racusen LC, et al. Banff ’05 Meeting Report: differential diagnosis of chronic allograft injury and elimination of chronic allograft nephropathy (“CAN”). Am J Transplant 2007;7:518e26. [15] Haas M. The significance of C4d staining with minimal histologic abnormalities. Curr Opin Organ Transplant 2010;15:21e7.