The acute rejection with intimal arteritis (ARV) is commonly considered as a severe form of acute rejection characterized by infiltration of mononuclear cells beneath the endothelium or by the presence of arteritis and is traditionally categorized into T cell–mediated rejection with v lesions (TCMRV) and antibody-mediated rejection with v lesions (AMRV) based on the presence of donor-specific HLA antibodies (DSA) and C4d staining.1 The AMR is supposed to relate to the poorer initial responses to antirejection treatment, allograft function and graft outcome compared to the TCMR.2-4 Over the years, the description of AMR has evolved based on continuous new research, and more areas are being investigated for new clarification.5 Recently, the consensus criteria of AMR have been revised and published in the 2013 Banff meeting report6; the coexistence of 3 features is required for the diagnosis of AMR: (1) the histological evidences of acute or chronic tissue injury; (2) the evidence of current/recent antibody interaction with vascular endothelium; (3) the serologic evidence of the circulating DSA; moreover, 2 important criteria are firstly included: (i) the C4d-positive staining is no longer the unique evidence of feature 2, which can also be indicated as positive by the presence of moderate microvascular inflammation (MI) lesions when C4d staining is negative; (ii) the biopsies contain only 2 features of AMR may be designated as suspicious AMR. We retrospectively studied 148 ARV episodes and defined the v lesion plus DSA and C4d staining or moderate MI lesions as AMRV, v lesion plus DSA or C4d staining or moderate MI lesions as sAMRV, v lesion alone and free of DSA, C4d staining and moderate MI lesions as TCMRV. We examined if the category and severity of ARV is relevant to distinct short-term (reversibility of the rejection episode with antirejection therapy) and long-term clinical outcome (graft survival).
MATERIALS AND METHODS
Patient and Data Collection
We retrospectively analyzed all adult (≥18 years) patients who received kidney transplant between 1996 and 2012 at the Kidney and Transplant Centre of Charité Campus Mitte, and had at least 1 for-cause graft biopsy that demonstrated histological features of ARV. In case 1 patient experienced multiple categories of ARV, the AMRV were preferentially chosen, followed by sAMRV and TCMRV. In addition, Banff v1, v2, and v3 lesions were defined as the low, moderate, and high ARV severity, respectively. In case there were multiple ARV episodes in 1 ARV category, v3-ARV was firstly chosen, followed by v2- and v1-ARV. All clinical and laboratory data were recorded in our transplant database system (TBase) at each visit. This study was approved by the institutional review committee.
Histopathology and C4d Staining
An ultrasound-guided graft biopsy was performed when clinically indicated, that is, elevated serum creatinine (Scr). All patients with delayed graft function, defined as needing dialysis post kidney transplantation,7 underwent protocol biopsy on the 7-day transplantation. A representative biopsy involved at least 1 artery and more than 7 glomeruli; all histological slides were examined by 2 pathologists (B.R. and W.K.), and all Banff scored lesions were assessed.1,4,6,8,9 The deposition of C4d was detected by indirect immunofluorescence on paraffin sections of formalin-fixed tissue (polyclonal anti-C4d antibody, Dianovo, Germany); more than 1% peritubular capillaries with linear deposition of C4d was defined as the cutoff for positivity. Biopsies from the pre-C4d era were retrospectively tested for C4d. The moderate MI were defined as glomerulitis (g)+ peritubular capillaritis (ptc) of 2 or higher, but the presence of g 1 or higher was required in the presence of acute TCMR, borderline infiltrates or evidence of infection.
HLA Antibody Screening
Each patient with ARV episode was tested for serum alloantibody; the DSA level was monitored as previously described.10 All serum samples which were collected once a year or at biopsy were qualitatively screened for HLA antibodies by 2 enzyme-linked immunosorbent assay–based screening systems (PRA-STAT and LAT) from 1996 to 2006 or the Luminex-based bead assay LABScreen Mixed (One Lambda, Canoga Park, CA) from 2007 onward. All tests were performed according to the manufacturer’s guidelines.11
Immunosuppression and Antirejection Treatment
The immunosupressive protocol comprised Cyclosporine A/Tacrolimus, mycophenolate mofetil/Azathioprine, and methylprednisolone. The doses of Cyclosporine A and Tacrolimus were adjusted according to whole blood trough levels. Antirejection therapy involved 2 broad steps: (1) pulse therapy of corticosteroids; (2) patients with steroid-resistant ARV or DSA received therapeutic PPH plus antibody therapy, which included 1 or more of following reagents: rabbit antithymocyte globulin; intravenous immune globulin; Rituximab (anti-CD 20 globulin); Bortezomib (therapeutic proteasome inhibitor); and Eculizumab (anti-Complement protein 5). Complete, partial and nonreversible responses were defined by Gaber et al12 by comparing the 1 month post biopsy Scr with the pre-biopsy concentration.
All data were assessed for completeness by a single investigator (S.D.). Continuous variables were expressed as mean± standard deviation. Categorical variables were expressed as N and percentage of total. Student t test was used to compare 2 groups of continuous variables and χ2 for categorical data. Death-censored graft failure, defined as returning to chronic dialysis, was analyzed by Kaplan-Meier graphs. To test putative risk factors for long-term graft loss, the influence of clinical variables and Banff lesions on graft survival were tested by univariate Cox-regression. The HLA antibody status, presence of delayed graft function, Banff scores of transplant glomerulopathy (cg), g, arteriolar hyaline, vascular intimal fibrosis, interstitial fibrosis/ tubular atrophy, mesangial matrix, and C4d staining were analyzed as binary variables (score = 0 vs. score >0). We included all variables with a P value less than 0.05 in the univariable analysis into multivariate analysis. All statistics were performed by using SPSS16.0 (SPSS Inc., Chicago, IL). P value less than 0.05 was considered significant.
Between January 1996 and December 2012, 1263 patients received only kidney transplantation and followed up for at least 6 months in our center. After excluding 1115 patients based on the criteria detailed in Figure S1 (SDC,http://links.lww.com/TP/B126), 148 ARV episodes were chosen from 148 patients and categorized into: TCMRV (n = 78, 52.7%) and total AMRV (n = 70, 47.3%), which were further divided into sAMRV (n = 37, 25.0%) and AMRV (n = 33, 22.3%).
Patient Demographics and Clinical Characteristics at the Time of Biopsy
There was no significant difference in recipient and transplant characteristics among the 3 ARV groups (Table 1). The median biopsy timing of AMRV was statistically longer than that of TCMRV (217 vs 12.5 days after transplantation, P = 0.01). The mean Scr concentration (pre-, at, and post-AVR) were comparable among the 3 AVR groups. Significantly more patients of sAMRV and AMRV received therapeutic PPH and antibodies therapy compared to TCMRV (each pairwise comparison to TCMRV group yields P < 0.01). However, no significant differences of the responses to the antirejection therapy were found among the 3 ARV groups. In the aspect of the ARV severity, significantly more patients with v2-ARV and v3-ARV received therapeutic PPH and antibody therapy compared to patients with v1-ARV (each pairwise comparison to v1-ARV group yields P < 0.01). The grafts with v1-ARV showed a significantly higher proportion of complete reversibility than that of v3-ARV (56.2% vs. 23.5%, P = 0.02); the v2- and v3-ARV presented with a significantly higher fraction of nonreversibility than that of v1-ARV (each pairwise comparison to v1-ARV yields P < 0.05), but comparable responses between v2-AVR and v3-AVR.
Histological Evaluation of the For-Cause Biopsies After Transplantation
As shown in Table 2, 67.9% TCMRV, 37.8% sAMRV, and 16.2% AMRV showed v1 lesion (overall P = 0.001, each pairwise comparison to TCMRV yields P < 0.05); in contrast, 3.8% TCMRV, 16.2% sAMRV, and 24.2% AMRV contained v3 lesion (P = 0.003, each pairwise comparison to TCMRV yields P < 0.05). Among 3 ARV groups, the AMRV group showed statistically higher grade of g, v, ptc, and mesangial matrix lesions compared to the TCMRV group (each pairwise comparison yields P < 0.01); the statistically higher grade of g lesion and C4d staining occurred in the AMRV group compared to the sAMRV group (each pairwise comparison yields P < 0.05). Patients of TCMRV group (21.9%) underwent single episode of for-cause biopsy compared to 9.1% of AMRV group (P = 0.03), and 42.3% patients of TCMRV group underwent single episode of biopsy-proven acute rejection (BPAR) compared to 18.2% of AMRV group (P = 0.006); in contrast, 30.3% patients of AMRV group experienced 3 BPAR episodes or more compared to 11.5% of TCMRV group (P = 0.008). Among v1-, v2-, and v3-ARV, 11.8% patients of sAMRV group underwent single episode of for-cause biopsy compared to 9.1% of AMRV group (P = 0.04), and 42.5% patients of TCMRV group underwent single episode of BPAR compared to 13.7% of sAMRV group (P = 0.008).
Impact of ARV Category on the Graft Outcomes
The 8-year death-censored graft survival (DCGS) rate was 56.8% of TCMRV and 34.1% of total AMRV (Log rank, P = 0.03); but the 1- and 5-year DCGS rates were comparable between TCMRV and total AMRV. The 1-, 5- and 8-year patient survival rates were comparable among the 3 ARV groups (each pairwise comparison between the 2 ARV groups yields P > 0.05, Figure 1A and B). In addition, the DCGS rates were similar between sAMRV and AMRV at 1, 5, and 8 years after transplantation.
Impact of ARV Severity on the Graft Outcomes
As shown in Figure 2A, the grafts with v2- and v3-ARV showed significantly lower DCGS rates than that of v1-ARV at 1, 5, and 8 years after transplantation (each pairwise comparison to v1-ARV yields P < 0.05), and the DCGS rate of v3-ARV was statistically lower than v2-ARV at 5 years after transplantation (P = 0.03). As shown in Table 3, part A, in the TCMRV group, the 8-year DCGS rates of grafts with v1, v2, and v3 lesions were 63.6%, 40.0%, and 0.0%, respectively (the pair-wise comparison between v1- and v3-TCMRV, yields P < 0.05); in the sAMRV group, the 8-year DCGS rate of grafts with v3 lesions was significantly lower than those with v1 lesion (P = 0.01); in the AMRV group, the 8-year DCGS rates of grafts with v2 and v3 lesions were significantly lower than those with v1 lesions (each pairwise comparison to v1-AMRV yields P < 0.05). In overall and each category, the grafts with the same grade of v lesion showed similar 8-year DCGS rate.
Impact of DSA Status on the Graft Outcomes
The 1-, 5- and 8-year DCGS rates were similar between overall C4d− ARV and C4d+ ARV (each pairwise comparison yields P > 0.05, Figure 3A); within 3 categories, the 8-year DCGS rate was comparable when the comparison was performed between the grafts in the presence and absence of DSA (Table 3, part B).
Impact of Moderate Microvascular Inflammation on the Graft Outcomes
The 1-, 5- and 8-year DCGS rates were similar between the overall ARV in the presence and absence of moderate MI lesions (each pairwise comparison yields P > 0.05, Figure 3C); as shown in Table 3, part B, within sAMRV and AMRV groups, the grafts with MI less than 2 or MI of 2 or higher showed similar 8-year DCGS rates (each pairwise comparison yields P > 0.05).
Impact of C4d Status on the Graft Outcomes
The 1-, 5- and 8-year DCGS rates were similar between overall C4d− ARV and C4d+ ARV (each pairwise comparison yields P > 0.05, Figure 3B). As shown in Table 3, part B, the AMRV with C4d 0 showed significantly lower DCGS rates than that of TCMRV with C4d 0 at 8 years after transplantation (0.0% vs 56.8%, P < 0.05).
Impact of the Tubulointerstitial Inflammation on the Graft Outcomes
32 biopsies showed minimal tubulointerstitial inflammation (TI) (Banff t ≤ 1 and i ≤ 1), the remaining biopsies presented intensive TI (t ≥ 2 or i ≥ 2); the 1-, 5-, and 8-year DCGS rates were similar between the overall grafts with minimal TI and with intensive TI (Figure 3D); in addition, the 8-year DCGS rates were comparable between grafts with minimal TI and with intensive TI in each category (Table 3, part B).
Analysis of Histological and Clinical Factors Associated With Graft Failure
We assessed the association of histological Banff lesions, HLA antibody status, and clinical variables with graft survival by univariate and multivariate analysis (Table S1, SDC,http://links.lww.com/TP/B126). By multivariate analysis, the presence of ptc lesions and DSA class I appeared to be independent factors inversely related to graft loss.
One hundred forty-eight patients who experienced ARV episodes in kidney transplants were reviewed and divided into AMRV, sAMRV, and TMRV groups. Significantly higher proportion of patients in sAMRV and AMRV groups received aggressive immunotherapies to manage alloantibodies; however, the 3 ARV groups presented similar responses to antirejection treatment. In addition, the patients of TCMRV group had relatively higher DCGS rates at 1, 5 years after transplantation than those of the total AMRV groups; albeit no difference reaches the statistical significance; even the 8-year DCGS rates were statistically higher in TCMRV group comparing to total AMRV group, but the difference was marginally significant (P = 0.03). Our data were in accordance with a recent study of Lefaucheur et al,13 who reported that the risk of graft loss in AMRV was not significantly increased in TCMRV at 6-year of transplantation. On one hand, the increased reversibility of corticosteroid resistant ARV is likely explained by the efficacy of the intensive immunosuppressive therapy direct on antibody production in addition to steroids and plasmapheresis; on the other hand, the 3 ARV categories which are reclassified according to the revised Banff criteria of AMR are not mutually exclusive and can be coexisted because the late onset or severe form of TCMR was often reported in conjunction with unrecognized AMR.14 Even we have excluded any biopsy showing positive DSA or C4d staining or moderate/severe MI lesions from TCMRV group, it was impossible to completely rule out the mixture AMR because the C1q-fixing DSA15 or non-HLA antibodies or the multilayering of the peritubular capillary basement membrane which is only evidence in electron microscopy16 have not been routinely checked.
In aspect of the ARV severity, defined by Banff scores of v lesion, it is proven as an independent histological factor associated with poor responses to the treatment and inferior prognosis. In our study, the grafts with v2- and v3-ARV were more often treated by the therapeutic plasmapheresis and antibody-directed therapy; however, the fraction of nonreversibility was still evidently higher compared to v1-ARV; conversely, ARV with the same grade of v lesion presented with similar responses to antirejection treatment regardless of the ARV categorization. The less reversibility of high grade v lesions might lead to more clinically for-cause biopsies and higher risk of subsequent late rejection and result in persistent and progressive renal parenchymal damage and acceleration of the graft failure.17,18 In addition, the high grade v lesion often coexisted with intimal fibrinoid necrosis, which responded very poor to antirejection therapy and most of patients lost grafts within a year.19 Furthermore, the infiltration of vessels by mononuclear cells can induce endothelial cell apoptosis, intimal fibroproliferation, and neointimal thickening; contribute to the progression of transplant arteriosclerosis (vascular intimal fibrosis lesion); and eventually develop into a luminal obliterative vasculopathy, a hallmark of the chronic allograft dysfunction in the long term.20,21
Given commonly concerned as an underlying humoral compartment, the circulating DSA can directly induce endothelial cell injury through the complement-dependent or complement-independent pathways, and has been proven to compromise the renal allograft survival.22-25 The recently revised Banff classification highlights the diagnostic importance of the serologic DSA and the endothelial injuries initiated from DSA, which can help to distinguish patients with AMR from other causes in the posttransplant setting. Therefore, testing for DSA is recommended in all biopsies from patients who have documented DSA at any time after transplantation and/or had a previous biopsy showing C4d staining, g, and/or peritubular capillaritis.26 In our study, DSA class I are an independent risk factor associated with the graft loss; however, the grafts with DSA+ ARV had relative poorer long-term graft outcome compared to the grafts with DSA− ARV. Our data suggest that the management of DSA by combined agents which target directly on removing the DSA and inhibiting the interaction of DSA to the endothelial cells might reduce the inferior impacts of AMR and improve the clinical outcomes.
Traditionally, the C4d staining is required for the diagnosis of AMR and strongly correlated with allograft loss27; however, in the revised Banff classification, the C4d staining is no longer specific for AMR due to its low sensitivity. In our study, the overall C4d-positive and -negative ARV showed similar long-term graft outcomes. The positive C4d staining commonly contributes to rejection as an underlying humoral component, thus an aggressive immunotherapy, such as antilymphocytic agents, will be given to improve the clinical outcomes; in addition, for C4d to be solely responsible for graft dysfunction, diffuse deposition is still required.28
The moderate MI lesions are considered as a remark of AMR in the revised Banff classification and the microcirculation endothelial injury has been previously proven to be a negative prognostic feature in late biopsies independent of C4d staining29,30; however, MI lesions exist not only in AMR but also in TCMR, the latter in absence with DSA has good graft prognosis.30 In our study, the grades of microcirculation endothelial injury (g and peritubular capillaritis) were significantly higher in AMRV group compared to the TCMRV group; the ARV with moderate MI lesions prognoses a similar long-term graft survival with the ARV with free or mild MI lesions, which suggest that the inferior impact of MI lesions on the allograft outcome is independent on the extent of MI lesions.
Moreover, the grafts with v lesion and one other feature of humoral components are defined as sAMRV in our study, which shows similar characteristics, clinical courses, and long-term graft outcome compared to the grafts with AMRV. It is possible that sAMRV and AMRV might represent a spectrum of the same disorder, not 2 different types of ARV with distinct pathophysiological states; the ARV initially presented as sAMR to the kidney allografts in the biopsy, but subsequently progressed over time to manifest AMR, this opinion has to be identified by follow-up biopsies in patients with unsuccessfully treated AMR to access the 3 features of AMR.
In conclusion, the severity of ARV is robustly associated with over time renal allograft dysfunction despite of the impacts of the humoral/cellular rejection components. So far, the ideal treatment of the 3 grades of v lesions still remains to be determined, but a more aggressive attempt to treat and prevent the high severity of ARV will be an effective strategy and a real impetus for improving kidney survival. In addition, the distribution of v lesions is not even, the optimal biopsy probes which contain at least 2 interlobular arteries will be highly recommended for the histological evaluation.
Partial data of this study has been presented on 26th European Congress of Pathology congress.
1. Racusen LC, Solez K, Colvin RB, et al. The Banff 97 working classification of renal allograft pathology. Kidney Int
. 1999; 55: 713–723.
2. Mueller A, Schnuelle P, Waldherr R, et al. Impact of the Banff ’97 classification for histological diagnosis of rejection on clinical outcome and renal function parameters after kidney transplantation. Transplantation
. 2000; 69: 1123–1127.
3. Nankivell BJ, Borrows RJ, Fung CL, et al. The natural history of chronic allograft nephropathy. N Engl J Med
. 2003; 349: 2326–2333.
4. Nankivell BJ, Alexander SI. Rejection of the kidney allograft. N Engl J Med
. 2010; 363: 1451–1462.
5. Haas M, Sis B, Racusen LC, et al. Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions. Am J Transplant
. 2014; 14: 272–283.
6. Sis B, Mengel M, Haas M, et al. Banff ’09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups. Am J Transplant
. 2010; 10: 464–471.
7. Huber L, Lachmann N, Dür M, et al. Identification and therapeutic management of highly sensitized patients undergoing renal transplantation. Drugs
. 2012; 72: 1335–1354.
8. Mengel M, Sis B, Haas M, et al. Banff 2011 Meeting report: new concepts in antibody-mediated rejection. Am J Transplant
. 2012; 12: 563–570.
9. Shimizu T, Tanabe T, Shirakawa H, et al. Acute vascular rejection after renal transplantation and isolated lesion. Clin Transplant
. 2012; 26: 2–8.
10. Liefeldt L, Brakemeier S, Glander P, et al. Donor-Specific HLA antibodies in a cohort comparing everolimus with Cyclosporine after kidney transplantation. Am J Transplant
. 2012; 12: 1192–1198.
11. Gaber LW, Moore LW, Alloway RR, et al. Correlation between Banff classification, acute renal rejection scores and reversal of rejection. Kidney Int
. 1996; 49: 481–487.
12. Lefaucheur C, Loupy A, Vernerey D, et al. Antibody-mediated vascular rejection of kidney allografts: a population-based study. Lancet
. 2013; 381: 313–319.
13. Matas AJ. Acute rejection is a major risk factor for chronic rejection. Transplant Proc
. 1998; 30: 1766–1768.
14. Yabu JM, Higgins JP, Chen G, et al. C1q-fixing human leukocyte antigen antibodies are specific for predicting transplant glomerulopathy and late graft failure after kidney transplantation. Transplantation
. 2011; 91: 342–347.
15. Miura M, Ogawa Y, Kubota KC, et al. Donor-specific antibody in chronic rejection is associated with glomerulopathy, thickening of peritubular capillary basement membrane, but not C4d deposition. Clin Transplant
. 2007; 21: 8–12.
16. Meier-Krische HU, Schold JD, Sirinivas TR, et al. Lack of improvement in renal allograft survival despite a marked decrease in acute rejection rates over the most recent era. Am J Transplant
. 2004; 4: 378–383.
17. Salmela KT, von Willebrand EO, Kyllonen LEJ, et al. Acute vascular rejection in renal transplantation-diagnosis and outcome. Transplantation
. 1992; 54: 858–862.
18. Haas M, Kraus ES, Samaniego-Picota M, et al. Acute renal allograft rejection with intimal arteritis: histologic predictors of response to therapy and graft survival. Kidney Int
. 2002; 61: 1516–1521.
19. Hillebrands JL, Rozing J. Chronic transplant dysfunction and transplant arteriosclerosis: new insights into underlying mechanisms. Expert Rev Mol Med
. 2003; 5: 1–23.
20. Ashman N, Chapagain A, Dobbie H, et al. Belatacept as maintenance immunosuppression for postrenal transplant de novo drug-induced thrombotic microangiopathy. Am J Transplant
. 2009; 9: 424–427.
21. Lefaucheur C, Loupy A, Hill GS, et al. Preexisting donor-specific HLA-antibodies predict outcome in kidney transplantation. J Am Soc Nephrol
. 2010; 21: 1398–1406.
22. Wiebe C, Gibson IW, Blydt-Hansen TD, et al. Evolution and clinical pathologic correlations of de novo donor-specific HLA antibody post kidney transplant. Am J Transplant
. 2012; 12: 1157–1167.
23. Zhang X, Reed EF. Effect of antibodies on endothelium. Am J Transplant
. 2009; 9: 2459–2465.
24. Valenzela NM, Hong L, Shen XD, et al. Blockade of P
-selectin is sufficient to reduce MHC I antibody-elicited monocyte recruitment in vitro and in vivo. Am J Transplant
. 2013; 13: 299–311.
25. Sis B, Campbell PM, Mueller T, et al. Transplant glomerulopathy, late antibody-mediated rejection and the ABCD tetrad in kidney allograft biopsies for cause. Am J Transplant
. 2007; 7: 1743–1752.
26. Ferucht HE, Schneebergr H, Hillebrand G, et al. Capillary deposition of C4d complement fragment and early renal graft loss. Kidney Int
. 993; 43: 1333–1338.
27. Magil AB, Tinckam KJ. Focal peritubular capillary C4d deposition in acute rejection. Nephrol Dial Transplant
. 2006; 21: 1382–1388.
28. Fahim T, Bohmig GA, Exner M, et al. The cellular lesion of humoral rejection: predominant recruitment of monocytes to peritubular and glomerular capillaries. Am J Transplant
. 2007; 7: 385–393.
29. Papadimitriou JC, Drachenberg CB, Munivenkatappa R, et al. Glomerular inflammation in renal allografts biopsies after the first year: cell types and relationship with antibody-mediated rejection and graft outcome. Transplantation
. 2010; 90: 1478–1485.
30. Sis B, Jhanri GS, Riopel J, et al. A new diagnostic algorithm for antibody-mediated microcirculation inflammation in kidney transplants. Am J Transplant
. 2012; 12: 1168–1179.
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