补体裂解片断C4d在移植肾急性排斥反应中的临床意义

目的探讨肾小管周围毛细血管补体裂解片断(C4d)沉积在移植肾急性排斥反应中的临床意义。方法对肾移植后发生急性排斥反应的78例受者进行移植肾活体穿刺检查,共获取移植肾活检穿刺标本86份。根据Banff97病理分型将86份活检标本分为BanffⅠ型32份,Ⅱ型51份,Ⅲ型3份。应用免疫组织化学法检测出86份标本中有30份出现肾小管周围毛细血管C4d沉积,阳性率为34.9%。分析C4d阳性其与Banff97分型、术前一般情况、抗排斥治疗、移植肾功能及移植肾预后的关系。结果BanffⅠ和Ⅱ型受者移植肾中C4d阳性率分别为21.9%和39.2%,两者相比差异无统计学意义(P=0.101)。有妊娠史、术前群体反应性抗体(PRA)>30%和再次移植的受者C4d阳性率较高。C4d阳性的受者发生排斥反应时血肌酐较阴性受者高,分别为(312.56±196.26)μmol/L和(210.97±136.59)μmol/L,两组差异有统计学意义(P=0.0115)。C4d阳性受者对激素和ATG冲击治疗与阴性受者比较,敏感率明显降低。C4d阳性的受者移植肾1年生存率较阴性受者低,分别为64.3%和90.0%,两组间差异有统计学意义(P=0.006)。结论移植肾C4d阳性的受者发生排斥反应时,对常规的激素冲击和ATG抗排斥治疗不敏感,血肌酐明显升高,移植肾1年存活率下降,受者预后较差。     

C4d complement split product expression in chronic rejection of renal allograft

Chronic allograft rejection remains the major cause of late renal graft loss. Its pathogenesis is complex, depending on both immunological and nonimmunological factors. An important role in development of chronic rejection is ascribed to an ongoing immunological reaction mainly of the humoral type. C4d complement split product, as a stable fragment of complement degradation activated by antigen-antibody complexes, is considered to be an indicator of humoral activity in allografts. The aim of the present study was to establish a correlation between C4d expression and morphological findings specific for chronic rejection among biopsy specimens from patients with deteriorating graft function versus protocol biopsy specimens versus biopsy specimens of native kidneys with glomerular diseases. C4d deposits in peritubular capillaries and glomeruli were observed in 83% of patients with morphological changes of chronic rejection. No C4d expression was found in the protocol biopsy group. C4d deposits in glomeruli localizations were found in kidneys from patients with glomerulopathies; the pattern of distribution was similar to that for antibodies characteristic for glomerulonephritis. There was a positive correlation between C4d expression and morphological features of chronic rejection. In our opinion, only peritubular capillary localization is specific for a rejection process; glomerular localization is nonspecific and probably secondary to antigen-antibody complex deposition in course of some types of glomerulopathies.     

Chronic humoral rejection: identification of antibody-mediated chronic renal allograft rejection by C4d deposits in peritubular capillaries

The pathogenesis of chronic renal allograft rejection (CR) remains obscure. The hypothesis that a subset of CR is mediated by antidonor antibody was tested by determining whether C4d is deposited in peritubular capillaries (PTC) and whether it correlates with circulating antidonor antibodies. All cases (from January 1, 1990, to July 31, 1999) that met histologic criteria for CR and had frozen tissue (28 biopsies, 10 nephrectomies) were included. Controls were renal allograft biopsies with chronic cyclosporine toxicity (n = 21) or nonspecific interstitial fibrosis (n = 10), and native kidneys with end-stage renal disease (n = 10) or chronic interstitial fibrosis (n = 5). Frozen sections were stained by two-color immunofluorescence for C4d, type IV collagen and Ulex europaeus agglutinin I. Antidonor HLA antibody was sought by panel-reactive antibody analysis and/or donor cross matching in sera within 7 wk of biopsy. Overall, 23 of 38 CR cases (61%) had PTC staining for C4d, compared with 1 of 46 (2%) of controls (P < 0.001). C4d in PTC was localized at the interface of endothelium and basement membrane. Most of the C4d-positive CR tested had antidonor HLA antibody (15 of 17; 88%); none of the C4d-negative CR tested (0 of 8) had antidonor antibody (P < 0.0002). The histology of C4d-positive CR was similar to C4d-negative CR, and 1-yr graft survival rates were 62% and 25%, respectively (P = 0.05). Since August 1998, five of six C4d-positive CR cases have been treated with mycophenolate mofetil +/- tacrolimus with a 100% 1-yr graft survival, versus 40% before August 1998 (P < 0.03). These data support the hypothesis that a substantial fraction of CR is mediated by antibody (immunologically active). C4d can be used to separate this group of CR from the nonspecific category of chronic allograft nephropathy and may have the potential to guide successful therapeutic intervention.     

Monocytes and peritubular capillary C4d deposition in acute renal allograft rejection

BACKGROUND: Peritubular capillary (PTC) deposition of complement split factor C4d in renal allografts has been shown to be closely associated with circulating antidonor antibodies and a marker for relatively poor graft survival. Monocyte/macrophage (MO) infiltration of renal allografts has been shown to adversely affect graft survival. The purpose of this study was to assess whether the two phenomena are related. METHODS: Twenty-three biopsies (from 15 patients) demonstrated diffuse strong staining of PTC for C4d (C4d+ group) and acute tubular injury with or without significant cellular rejection, while 28 biopsies (with acute rejection) but negative for PTC C4d served as controls (C4d- group). RESULTS: The C4d+ group demonstrated significantly greater glomerular and interstitial MO infiltration than did the C4d- group [3.4 +/- 2.0 vs. 0.2 +/- 0.3 MO/glomerulus, P < 0.0001; 12.9 +/- 9.2 vs. 6.5 +/- 5.0 MO/high power field (hpf), P = 0.0030]. Neutrophilic (PMN) infiltration of glomeruli and PTC was also significantly greater in the C4d+ group than in the C4d- one (0.8 +/- 0.6 vs. 0.3 +/- 0.3 PMN/glomerulus, P = 0.0003; 0.9 +/- 0.8 vs. 0.4 +/- 0.3 PTC PMN/hpf, P = 0.0035). CONCLUSION: The results indicate a close association between PTC C4d deposition and MO infiltration, particularly glomerular, and confirm previous observations regarding the correlation of PTC C4d staining and PMN infiltration.     

Complement activation in acute humoral renal allograft rejection: diagnostic significance of C4d deposits in peritubular capillaries.

The distinction between acute humoral rejection (AHR) and acute cellular rejection (ACR) in renal allografts is therapeutically important, but pathologically difficult. Since AHR is probably mediated by antibodies to the donor endothelium that activate the classical complement pathway, it was hypothesized that peritubular capillary C4d deposition might distinguish this group. Renal biopsies (n = 16) from 10 patients with AHR who had acute graft dysfunction, neutrophils in peritubular capillaries, and a concurrent positive cross-match were stained for C4d by immunofluorescence. Control biopsies for comparison showed ACR (n = 14), cyclosporin A toxicity (n = 6), or no abnormality (n = 4). Peribiopsy sera were tested for anti-donor HLA antibody. C4d deposited prominently and diffusely in the peritubular capillaries in all AHR biopsies (16 of 16). IgM and/or C3 were also present in 19 and 44%, respectively. With two-color immunofluorescence, C4d was localized in basement membranes (type IV collagen+) and in the endothelium (Ulex europaeus agglutinin-I+). In ACR, no more than trace C4d was found in peritubular capillaries (P < 0.0001 versus AHR), and no patient had anti-donor HLA antibodies (0 of 8); 27% had neutrophils in peritubular capillaries. One of six biopsies with cyclosporin A toxicity had similar C4d deposits, and circulating anti-donor class I antibody was detected. Grafts with AHR were lost (40%) more often than those with ACR (0%; P < 0.02). C4d in peritubular capillary walls distinguishes AHR from ACR, is more specific and sensitive than traditional criteria, and is a potentially valuable adjunct in the diagnosis of graft dysfunction.     

Donor-specific antibody in chronic rejection is associated with glomerulopathy, thickening of peritubular capillary basement membrane, but not C4d deposition

Abstract:  The impact of post-transplant donor-specific antibody (DSA) on the development of chronic rejection has been focused recently. The aim of this study was to evaluate the significance of DSA, graft function and pathological factors of chronic rejection. Seventy-three kidney recipients who underwent protocol biopsy were included in the study. The median follow-up period after transplant was 40 months. The presence of anti-HLA antibody (aHLAAb) and DSA was tested using flow beads analysis (FlowPRA®). The patients were divided into a group with DSA, a group with non-donor-specific aHLAAb and a group without aHLAAb. Protocol biopsy specimen were compared for transplant glomerulopathy (cg), vasculopathy (cv), C4d deposition at peritubular capillary (PTC), peritubular capillaritis (ptc score 0–3) and thickening of PTC basement membrane (ptcbm score 0–3) as recently proposed. The presence of DSA was significantly associated with the presence of cg, ptcbm. The group with non-donor-specific aHLAAb had ptcbm but did not have cg. The group without aHLAAb rarely showed ptcbm. The presence of DSA was associated with impaired graft function. C4d was not specific for the patients with DSA. These histopathological markers are useful in the detection of immunological chronic rejection. Early detection by screening tests will be important for treatment before irreversible change occurs.     

Capillary deposition of complement C4d and C3d in pediatric renal allograft biopsies

BACKGROUND: Peritubular capillary deposition of C4d (C4d(PTC)) is a marker of antibody-mediated alloresponse and is associated with poor graft survival in adults. C3d(PTC) has received less attention; its significance is unclear. To date no information has been gained in children. METHODS: The prevalence of C4d(PTC) and C3d(PTC) in pediatric renal allograft biopsies (n=77, 31 cadaveric kidneys) was analyzed retrospectively. Associations with histology, donor-specific antibodies (DSAs), and outcome were investigated. RESULTS: The overall prevalence of C4d(PTC) and C3d(PTC) was 52% and 48%, respectively. C3d(PTC) was associated with C4d(PTC) (P<0.0001). Thirty-six percent of acute rejections were cellular, 28% were humoral, and 36% were combined cellular and humoral. C3d(PTC) was found in 57% of acute rejection biopsies. C4d(PTC), but not C3d(PTC), was associated with accumulation of polymorphonuclear cells in peritubular capillaries (P=0.02). Fifty-one percent of late biopsies (>6 months posttransplantation) had features of chronic allograft nephropathy: 50% were C4d(PTC_ positive, and 50% were C3d(PTC) positive. C4d(PTC) positive chronic allograft nephropathy biopsies had more transplant glomerulopathy (P=0.020) and mesangial matrix increase (P=0.026). C3d(PTC) tended to be associated with transplant glomerulopathy (P=0.06), but not with mesangial matrix increase. C4d(PTC) was correlated with DSA (P=0.011). Excluding early nonrejection graft losses, more grafts were lost in the C4d(PTC) positive group (P=0.019). C3d(PTC) was not associated with DSA or graft outcome. CONCLUSIONS: Our results support C4d(PTC) being a hallmark of humoral rejection in pediatric renal transplantation; its presence was associated with DSA and poorer immunologic graft outcome. In contrast, C3d(PTC), although highly associated with C4d(PTC), did not correlate with DSA or outcome.     

Capillary deposition of the complement fragment C4d in cardiac allograft biopsies is associated with allograft vasculopathy

Cardiac allograft vasculopathy (CAV) is a long-term threat in heart transplant recipients and its exact pathogenesis remains to be established. As complement activation contributes to early and late allograft dysfunction, we hypothesized that deposition of the complement fragment, C4d, in capillaries of cardiac allograft biopsies may be associated with CAV. A polyclonal anti-C4d antibody was used for immunohistochemistry on endomyocardial biopsies obtained from heart transplant recipients during the first year post-transplantation. CAV was assessed by intracoronary ultrasound performed at 1-year post-transplantation. We were able to show that CAV is highly associated with C4d deposition in capillaries of cardiac allografts and that serial C4d studies may predict development of CAV at 1-year post-transplantation.     

Implications of immunohistochemical detection of C4d along peritubular capillaries in late acute renal allograft rejection

BACKGROUND: Immunohistochemical detection of the C4d complement product along peritubular capillaries (PC) may indicate humoral rejection of renal allografts. We examined the frequency of PC C4d expression in renal-allograft biopsies with acute rejection (AR) arising more than 6 months after transplantation and the impact of this finding. METHODS: C4d was detected by immunoperoxidase in 2-micron paraffin sections of consecutive biopsies obtained over a 3-year period. The extent was classified as diffuse (> or =50% PC C4d+), focal (<50% C4d+), and negative (C4d-). Clinical data were obtained by retrospective chart review. Fifty-five AR episodes with Banff 97 types 1A (n = 13), 1B (n = 26), 2A (n = 11), 2B (n = 3), and 3 (n = 2) met inclusion criteria. RESULTS: PC C4d expression was diffuse in 23 (42%), focal in 9 (16%), and negative in 23 (42%) biopsies. AR episodes with focal and diffuse C4d expression had higher proportionate elevation of serum creatinine at biopsy and 4 weeks after diagnosis (P< or =0.05). Biopsies with diffuse PC C4d had interstitial hemorrhage (56.5%) and plasmacytic infiltrates (52%) more frequently than C4d- biopsies (22% and 16%), P = 0.02, but had no other distinctive histologic features. Graft loss was greater in diffuse (65%) compared with focal C4d+ (33%) and C4d- (33%) groups 1 year after diagnosis, P = 0.03. Other clinical and pathologic parameters did not differ significantly, including treatment received for AR. CONCLUSION: Evidence of acute cellular with occult humoral rejection is identified in more than 40% of late AR episodes. Late acute humoral rejection may be associated with interstitial hemorrhage and plasma cells and contributes significantly to graft loss.     

Clinicopathological relevance of granular C4d deposition in peritubular capillaries of kidney allografts

While linear C4d staining in peritubular capillaries (PTC) is established as a marker of antibody‐mediated rejection, the significance of a distinct granular C4d deposition pattern has not yet been clarified. In this study, 329 renal allograft recipients who underwent indication biopsies were analysed for immunohistochemical C4d staining characteristics. Fifty‐six (17%) recipients showed granular C4d in PTC, without any relationship to conventional risk factors and morphological features of rejection. We found a strong association with long‐term overall graft survival (7‐year survival: 41% vs. 66% in granular C4d‐negative subjects, P = 0.001), which was mainly driven by a greater risk of mortality [hazard ratio: 3.12 (95% confidence interval: 1.23–7.94); P = 0.02]. Granular C4d was associated with delayed graft function [39% vs. 22% (C4d‐negative subjects), P = 0.007], higher 1‐year serum creatinine [median 2.1 (interquartile range: 1.7–2.6) mg/dl vs. 1.6 (1.3–2.0) mg/dl, P = 0.001] and a trend towards worse death‐censored graft survival (P = 0.07). In support of a role of capillary immune complex formation, granular C4d was associated with electron‐dense deposits in PTC basement membranes, which were occasionally accompanied by focally distributed capillary IgG deposits. In conclusion, our study suggests clinical relevance of detecting capillary granular C4d deposition. Our results point to a pathogenetic role of alloimmune‐independent immune complex deposition.     

Complement fragment C4d deposition in peritubular capillaries in acute humoral rejection after ABO blood group-incompatible human kidney transplantation

BACKGROUND: Acute humoral rejection (AHR) is the most important risk factor for early graft loss in ABO-incompatible (ABO-i) kidney transplantation (RTx). The pathogenesis and diagnostic criteria for AHR after ABO-i RTx remain unclear. Complement fragment C4d deposition in peritubular capillaries (PTC), which is a sensitive indicator for activation of the classical complement pathway, was studied to establish the pathologic diagnostic indicator of AHR. METHODS: Forty-four graft biopsy specimens from 19 patients with ABO-i living donors were analyzed within 90 days after RTx. Nineteen biopsy specimens with acute rejection after ABO-compatible (ABO-c) living-related RTx were used as controls. Diffuse and bright C4d deposition in PTC was considered significantly positive. RESULTS: All of 8 recipients with AHR showed significantly positive C4d in PTC in the ABO-i group, but 9 of 11 recipients without AHR were negative. In the ABO-c RTx group, 16 of 19 recipients were negative for C4d in PTC. The prevalence of C4d in PTC was significantly higher in ABO-i RTx (P<0.05). CONCLUSIONS: C4d deposition is valuable as a specific and sensitive indicator for AHR, even of mild severity, in ABO-i RTx.     

C4d complement split product in diagnosis of immunological activity of chronic allograft nephropathy

INTRODUCTION: Activation of the humoral branch of the immunological response is currently believed to play an important role in pathogenesis of chronic allograft nephropathy. The impact of humoral alloreactivity, indicated by the presence of C4d deposits in peritubular capillaries of a renal allograft, on the development of chronic allograft nephropathy is a significant problem in transplantation. The aim of the study was to assess and correlate C4d expression in patients with chronic allograft nephropathy, with clinical and morphological variables, as well as to assess the impact of a change in immunosuppression regimen on posttransplant course and renal allograft morphology. PATIENTS AND METHODS: Twenty-six patients with chronic allograft nephropathy underwent biopsies to correlate C4d expression with clinical parameters and morphological findings. In all patients azathioprine was replaced with mycophenolate mofetil with additional CsA dose reduction in 12 patients. After 1 year, 14 protocol biopsies were performed. RESULTS: The frequency of C4d peritubular capillary deposition among patients with chronic allograft nephropathy was 30%. C4d expression appeared later after transplantation, was correlated with chronic allograft glomerulopathy and proteinuria but not other clinical or histological variables. C4d deposits displayed no independent impact on serum creatinine level. Proteinuria was significantly more reduced in the C4d(+) group. Progression of chronic morphological changes was significantly accelerated in the C4d(+) group. CONCLUSION: C4d peritubular capillary expression did not differentiate patients after immunosuppression enhancement, but it predisposed to progression of chronic morphological findings during 1-year observation.     

C4d沉积在抗体介导的慢性排斥反应中的临床意义

Objective To explore the significance of peritubular capillary C4d deposition in histopathological changes, renal function and prognosis of the patients with antibody-mediated chronic rejection (AMCR). Methods Deposition of C4d in the kidney was examined by irnmunohistochemistry on routine paraffin-embedded sections using anti-C4d polyclonal antibody. Seventy-seven patients were divided into C4d+ group (n = 35) and C4d- group (n = 42). The relationship of C4d and renal function,histopathological changes and prognoses of allografts were analyzed. Results The number of patients with tubular atrophy and glomerular basement membrane proliferation in C4d+ group was significantly more than that in C4d group (P＜0.05). Mean serum creatinine level was significantly higher in C4d+ group than in C4d- group 12 months after renal transplantation [(379.1 + 260.2)μmol/L vs (260.5 + 175.3) μmol/L, P＜0.05]. According to Kaplan-Meier analysis, the one-year graft survival rate was lower in the C4d+ group (62.9% ) than in the C4d- group (83.3% ) (logrank P＜0.05). Conclusion Patients with C4d deposition are associated with tubular atrophy and glomerular basement membrane proliferation. The serum creatinine level in C4d+ patients was significantly higher than in C4d- group at the 12th month after transplantation. More patients with C4d deposition lost their grafts during the study period.     

C4d沉积在抗体介导的慢性排斥反应中的临床意义

目的 探讨移植肾肾小管周围毛细血管补体片段C4d沉积与抗体介导的慢性排斥反应的病理形态、移植肾功能及预后的关系.方法 应用免疫组织化学技术检测77例肾移植受者移植肾肾小管周围毛细血管中C4d的沉积情况,根据检测结果分为C4d阳性组(35例)和C4d阴性组(42例).检测并比较C4d阳性和阴性组受者移植肾的病理形态结构、移植肾功能及预后.结果 与C4d阴性组比较,C4d阳性组受者移植肾肾小管萎缩和肾小球基底膜增生分层的例数明显增多,差异有统计学意义(P＜0.05).C4d阳性组受者的血肌酐水平在移植肾穿刺后12个月时较C4d阴性组受者明显升高,分别为(379.1±260.2)μmol/L和(260.5±175.3)μmol/L,差异有统计学意义(P＜0.05).C4d阳性组受者移植肾穿刺明确C4d阳性后1年内移植肾存活率为62.9%(22/35),而阴性组受者为83.3%(35/42),两组比较,差异有统计学意义(P＜0.05).结论 在抗体介导的慢性排斥反应中,移植肾C4d沉积常见的病理学改变为肾小管萎缩和肾小球基底膜增生分层,C4d阳性抗体介导的体液性慢性排斥反应加快了移植肾功能丧失的进展速度,使C4d阳性受者的移植肾功能丧失率升高,导致存活率降低.     

C4d沉积在抗体介导的慢性排斥反应中的临床意义

Objective To explore the significance of peritubular capillary C4d deposition in histopathological changes, renal function and prognosis of the patients with antibody-mediated chronic rejection (AMCR). Methods Deposition of C4d in the kidney was examined by irnmunohistochemistry on routine paraffin-embedded sections using anti-C4d polyclonal antibody. Seventy-seven patients were divided into C4d+ group (n = 35) and C4d- group (n = 42). The relationship of C4d and renal function,histopathological changes and prognoses of allografts were analyzed. Results The number of patients with tubular atrophy and glomerular basement membrane proliferation in C4d+ group was significantly more than that in C4d group (P＜0.05). Mean serum creatinine level was significantly higher in C4d+ group than in C4d- group 12 months after renal transplantation [(379.1 + 260.2)μmol/L vs (260.5 + 175.3) μmol/L, P＜0.05]. According to Kaplan-Meier analysis, the one-year graft survival rate was lower in the C4d+ group (62.9% ) than in the C4d- group (83.3% ) (logrank P＜0.05). Conclusion Patients with C4d deposition are associated with tubular atrophy and glomerular basement membrane proliferation. The serum creatinine level in C4d+ patients was significantly higher than in C4d- group at the 12th month after transplantation. More patients with C4d deposition lost their grafts during the study period.     

Diffuse C4d staining of peritubular capillaries in renal allograft following bamlanivimab therapy

Neutralizing monoclonal antibodies such as bamlanivimab emerged as promising agents in treating kidney transplant recipients with COVID-19. However, the impact of bamlanivimab on kidney allograft histology remains unknown. We report a case of a kidney transplant recipient who received bamlanivimab for COVID-19 with subsequent histologic findings of diffuse peritubular capillary C4d staining. A 33-year-old man with end-stage kidney disease secondary to hypertension who received an ABO compatible kidney from a living donor, presented for his 4-month protocol visit. He was diagnosed with COVID-19 44 days prior to his visit and had received bamlanivimab with an uneventful recovery. His 4-month surveillance biopsy showed diffuse C4d staining of the peritubular capillaries without other features of antibody-mediated rejection (ABMR). Donor-specific antibodies were negative on repeat evaluations. ABMR gene expression panel was negative. His creatinine was stable at 1.3 mg/dl, without albuminuria. Given the temporal relationship between bamlanivimab and our observations of diffuse C4d staining of the peritubular capillaries, we hypothesize that bamlanivimab might bind to angiotensin-converting enzyme 2, resulting in classical complement pathway and C4d deposition. We elected to closely monitor kidney function which has been stable at 6 months after the biopsy. In conclusion, diffuse C4d may present following bamlanivimab administration without any evidence of ABMR.     

移植肾动脉内膜炎的细胞类型及其与管周毛细血管C4d沉积的关系

目的 研究同种异体肾移植术后急性血管性排斥患者动脉内膜炎的细胞类型及其与管周毛细血管（PTC）C4d沉积的关系。 方法 共纳入20例自2006年6月至2008年6月在本中心行同种异体肾移植手术后肾功能异常且病理证实为急性血管性排斥受者的21份肾活检组织。免疫组化方法检测同一活检标本连续切片内T细胞表面标记物CD3、巨噬细胞表面标记物CD68及体液性排斥特异性标记物C4d的表达情况。根据PTC C4d的沉积情况将患者分为C4d阳性（C4d+）组和C4d阴性（C4d-）组,定量计数中动脉内膜阳性细胞数,并计算每个中动脉横切面的平均细胞数。 结果 急性血管性排斥患者动脉内膜都以巨噬细胞浸润为主;C4d+组和C4d-组患者的动脉内膜都以巨噬细胞浸润为主,与浸润的T淋巴细胞数相比差异均有统计学意义[（12.45±9.86）个/ 中动脉横切面比（3.91±3.03）个/中动脉横切面,P = 0.007;（3.47±1.89）个/中动脉横切面比（1.45±1.37）个/中动脉横切面,P = 0.006],且C4d+组患者动脉内膜巨噬细胞浸润数量显著多于C4d-组（P = 0.007）。 结论 肾移植术后急性血管性排斥患者动脉内膜以巨噬细胞浸润为主,且与PTC C4d的沉积有相关性。C4d+患者动脉内膜巨噬细胞数显著多于C4d-患者。