Peritubular Capillary C4d Staining Research Articles

Early and Late Microvascular Inflammation Have Differing Etiological Causes and Clinical Expression.

Microvascular inflammation (MVI) is an important pathological feature of antibody-mediated rejection (AMR). How posttransplant time affects its clinicopathological expression is little understood. This retrospective, single-center study screened 3398 kidney transplant biopsies and dichotomized 202 MVI ≥ 2 (Banff glomerulitis + peritubular capillaritis ≥ 2) samples by 9-mo median incidence time for comparison. The prevalence of MVI ≥ 2 was 12.4% in transplant kidneys, which failed more frequently than propensity-matched normal controls (n = 202; P < 0.001). Epidemiological risk factors for early MVI ≥ 2 were delayed graft function, prior AMR, and circulating donor-specific antibodies (DSAs+). Prior recipient sensitization occurred in 72.3%. Early MVI ≥ 2 was classified AMR in 65.3% and cellular rejection in 34.7%, and demonstrated excellent functional recovery and graft survival comparable to normal control kidneys. Late MVI ≥ 2 was predicted by younger (18 = 29 y) age, female recipient, living-donation, prior methylprednisolone, cyclosporine (versus tacrolimus, levels <5 ng/mL), absent antiproliferative therapy, and DSA+ using multivariable epidemiological modeling. Nonadherence caused 49.5%, with iatrogenic minimization responsible for 47.5%, usually for recipient infection. Late MVI ≥ 2 was because of AMR in 93.1%, and characterized by greater interstitial fibrosis, tubular atrophy, complement degradation split-product 4d (C4d) staining of peritubular capillaries+, endothelial C4d staining of glomerular capillaries+, transplant glomerulopathy and vasculopathy scores, DSA strength, and graft failure than early MVI ≥ 2 or normal transplant kidneys. Death-censored graft survival in 149 unique MVI ≥ 2 kidneys was independently determined by nonadherence, serum creatinine, proteinuria, DSA+, Banff C4d staining of peritubular capillaries+, and chronic interstitial fibrosis scores. MVI score and time lost significance using multivariable Cox regression. The changing expression of MVI ≥ 2 over time is best explained by differences in underimmunosuppression and microvascular injury from AMR impacting allograft function and survival.

The relationship of microvascular inflammation with antibody-mediated rejection in kidney transplantation

Microvascular inflammation (MVI) is a key diagnostic feature of antibody-mediated rejection (AMR); however, recipients without donor-specific antibodies (DSA) defy etiologic classification using C4d staining of peritubular capillaries (C4dptc) and conventional DSA assignment. We evaluated MVI ≥ 2 (Banff g + ptc ≥ 2) using Banff 2019 AMR (independent of MVI ≥ 2 but including C4dptc) with unconventional endothelial C4d staining of glomerular capillaries (C4dglom) and - arterial endothelium and/or intima (C4dart) using tissue immunoperoxidase, shared-eplet and subthreshold DSA (median fluorescence intensity, [MFI] 100-499), and capillary ultrastructure from 3398 kidney transplant samples for evidence of AMR. MVI ≥ 2 (n = 202 biopsies) from 149 kidneys (12.4% prevalence) correlated with DSA+, C4dptc+, C4dglom+, Banff cg, i, t, ti scores, serum creatinine, proteinuria, and graft failure compared with 202 propensity score matched normal controls. The laboratory reported DSA− MVI ≥ 2 (MFI ≥500) occurred in 34.7%; however, subthreshold (28.6%), eplet-directed (51.4%), and/or misclassified anti-Human leukocyte antigen (HLA) DSA (12.9%) were identified in 67.1% by forensic reanalysis, with vascular C4d+ staining in 67.1%, and endothelial abnormalities in 57.1%, totaling 87.1%. Etiologic analysis attributed 62.9% to AMR (77.8% for MVI with negative reported DSA [DSA− MVI ≥2] with glomerulitis) and pure T cellular rejection in 37.1%. C4dptc−DSA− MVI ≥ 2 was unrecognized AMR in 48.0%. Functional outcomes and graft survival were comparable to normal controls. We concluded that DSA− MVI ≥ 2 frequently signified a mild “borderline” phenotype of AMR which was recognizable using novel serologic and pathological techniques.

Phosphorylation of S6RP in peritubular capillaries of kidney grafts and circulating HLA donor-specific antibodies.

Antibody-mediated rejection (ABMR) caused by donor-specific HLA-antibodies (DSA) is a mediator of allograft loss after kidney transplantation (KT). DSA can activate microvascular endothelium damage through the mTOR pathway. In this study we assessed the mTOR pathway activation by DSA in KT with ABMR (ABMR + DSA+) compared to controls (ABMR-DSA-), biopsies with ABMR changes without DSA (ABMR + DSA-) and DSA without ABMR changes (ABMR-DSA+), and the potential modulation by mTOR inhibitors (mTORi). We evaluated 97 biopsies: 31 ABMR + DSA+, 33 controls ABMR-DSA-, 16 ABMR + DSA-, and 17 ABMR-DSA+ cases. Regarding immunosuppression of full ABMR + DSA+ and controls, 21 biopsies were performed under mTORi treatment (11 of them ABMR + DSA+ cases) and 43 without mTORi (20 of them ABMR + DSA+) so as to explore its effect on the mTOR pathway. Biopsies were stained for C4d, Ki67, and phosphorylated (p) S6RP, ERK, and mTOR by immunohistochemistry. Labeling was graded according to peritubular capillary staining. ABMR biopsies showed significantly higher C4d, p-S6RP, and Ki67 staining in peritubular capillaries (PTC) compared to controls, and light differences in p-ERK or p-mTOR. mTORi treatment did not modify p-S6RP, p-mTOR, and p-ERK staining. Diffuse p-S6RP in PTC in the biopsies significantly associated with circulating HLA-DSA independently of graft rejection, and with worse death-censored graft survival. These findings suggest that activation of endothelium through the mTOR pathway evidence different mechanisms of damage in ABMR + DSA+ and ABMR + DSA- despite similar histological injury.

The determinants, biomarkers, and consequences of microvascular injury in kidney transplant recipients.

Independent of the initial cause of kidney disease, microvascular injury to the peritubular capillary network appears to play a central role in the development of interstitial fibrosis in both native and transplanted kidney disease. This association is explained by mechanisms such as the upregulation of profibrotic genes and epigenetic changes induced by hypoxia, capillary leakage, endothelial and pericyte transition to interstitial fibroblasts, as well as modifications in the secretome of endothelial cells. Alloimmune injury due to antibody-mediated rejection and ischemia-reperfusion injury are the two main etiologies of microvascular damage in kidney transplant recipients. The presence of circulating donor-specific anti-human leukocyte antigen (HLA) antibodies, histological findings, such as diffuse C4d staining in peritubular capillaries, and the extent and severity of peritubular capillaritis, are commonly used clinically to provide both diagnostic and prognostic information. Complement-dependent assays, circulating non-HLA antibodies, or evaluation of the microvasculature with novel imaging techniques are the subject of ongoing studies.

Histological picture of antibody-mediated rejection without donor-specific anti-HLA antibodies: Clinical presentation and implications for outcome.

In this cohort study (n=935 transplantations), we investigated the phenotype and risk of graft failure in patients with histological criteria for antibody-mediated rejection (ABMR) in the absence of circulating donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA), and compared this to patients with definite ABMR and HLA-DSA-positivity. The histological picture did not differ between HLA-DSA-positive (n=85) and HLA-DSA-negative (n=123) cases of ABMR histology, apart from increased complement split product 4d (C4d) deposition in the peritubular capillaries in HLA-DSA-positive cases. Histology of ABMR without HLA-DSA was more transient than DSA-positive ABMR, and patients with ABMR histology without HLA-DSA had graft survival superior to that of HLA-DSA-positive patients, independent of concomitant T cell-mediated rejection (38.2%) or borderline changes (17.9%). Multivariate analysis showed that the risk of graft failure was not higher in patients with histological picture of ABMR (ABMRh ) in the absence of HLA-DSA, compared to patients without ABMRh . Despite an association between C4d deposition and HLA-DSA-positivity, using C4d deposition as alternative for the DSA criterion in the diagnosis of ABMR, as proposed in Banff 2017, did not contribute to the prognosis of graft function and graft failure. We concluded that biopsies with ABMRh but without detectable HLA-DSA represent a distinct, often transient phenotype with superior allograft survival.

Concurrent Antibody-Mediated Rejection in Post-Transplant IgA Nephropathy – Diagnosis Using a Stepwise Approach of Graft Morphology

Background Diagnosis of concurrent antibody-mediated rejection (AMR) is difficult in cases of post-transplant IgA nephropathy (TxIgAN) with glomerular inflammation. Glomerular inflammatory cell infiltration may be seen either in the contexts of AMR or immune complex-mediated injury and described as ‘glomerulitis’ (‘G’) or ‘endocapillary hypercellularity’ (‘E’), respectively. Methods We analyzed 40 renal allograft biopsy samples of 37 TxIgAN patients having glomerular inflammatory cells (‘E’ and/or ‘G’) between January 1992 and December 2016, in search for ancillary morphologic features in the diagnosis of concurrent AMR. Results Glomerular inflammation was described as ‘G’, ‘E’, or both in 11, 14, and 15 biopsies, respectively. Morphologic features supportive of acute and chronic AMR (AAMR and CAMR, respectively), comprising peritubular capillaritis, glomerular basement membrane doubling, peritubular capillary basement membrane multilayering, and peritubular capillary C4d staining, were sequentially examined, which revealed nine AAMR and 14 CAMR. C4d was positive in 77.8% of AAMR and 42.9% of CAMR. Of the remaining 17 biopsy samples without AMR, mesangial hypercellularity and/or crescents were present in 14 samples. In four samples, acute T cell-mediated rejection coexisted. Conclusions Diagnosis of concurrent AMR can be aided by using a stepwise approach of morphologic features of AMR in cases of TxIgAN with glomerular inflammation.

Pattern of Glomerular Staining for C4d Immunohistochemistry is Useful for the Diagnosis of Immune Complex Mediated Glomerulonephritis in Renal Allograft Biopsies

Introduction Performance of the C4d stain is required for the adequate evaluation of kidney transplant biopsies (KTBx). Positive C4d staining in the renal peritubular capillaries is very useful for the diagnosis of antibody mediated allograft rejection (AMR) and correlates with poorer graft outcomes. Glomerular C4d staining is common in the chronic phases of AMR but this is not diagnostically useful due to inconsistent distribution and intensity of staining. Deposition of the C4d complement component in immune complex mediated (ICM) glomerulonephritis (GN) is expected but this has not been systematically studied in KTBx. Materials and Methods C4d stain (immunohistochemical method) was applied to 2432 consecutive, paraffin embedded KTBx, over a period of 5 years. In KTBx due to abrupt increase in proteinuria or history of GN in the native kidney, immunofluorescence and electron microscopy studies for standard evaluation of GN were also used for diagnosis. Results and Discussion In this unselected biopsy cohort, 33 KTBx from 27 patients showed recurrent or primary GN: 12 membranous glomerulopathy (MGN), 4 lupus nephritis (SLE), 9 IgA nephropathy and 8 focal segmental glomerulosclerosis (FSGS), Distinctive and consistent glomerular C4d stainining was noted in patients with MGN with a pattern paralleling the IF and EM findings. SImilarly, in recurrnet SLE, glomerular C4d staining corresponded to the histological lupus Class determined by light microscopy, IF and EM. The C4d staining pattern in these GNs was clearly different from that seen in transplant glomerulopathy. C4d staining in IgA nephropathy was inconsistent, mostly mesangial and did not correlate clearly with the strength of IF IgA staining. Only the segmental sclerotic lesions in FSGS were highlighted by the C4d staining in a manner that appeared nonspecific. Conclusions C4d staining is routinely evaluated in transplant biopsies and this stain consistently highlights immune complex deposits in MGN and SLE. Recognition of this distinctive staining pattern is helpful in the diagnosis of these processes and should prompt complete evaluation with IF and EM if these studies were not initially available. The findings in this study are based on C4d stain evaluation of paraffin embedded tissue sections. Since C4d staining corresponds to the classical pathway of complement activation, similar findings are expected with the IF staining method in fresh frozen tissue sections.

The prognostic values of caveolin-1 immunoreactivity in peritubular capillaries in patients with kidney transplantation.

The low sensitivity of C4d immunoreactivity in peritubular capillaries (PTCs) hinders its use in the diagnosis of chronic active antibody-mediated rejection (CAAMR). C4d-negative CAAMR was defined in the 2013 Banff classification, which included the expression of endothelial-associated transcripts (ENDATs). We previously showed that the ENDAT caveolin-1 (CAV-1) is a distinct feature of CAAMR. In this study, we investigated the prognostic value of CAV-1 immunoreactivity in PTCs in kidney transplant patients. Ninety-eight kidney transplant recipients were included in this study. The prognostic value of CAV-1 immunoreactivity in PTCs was evaluated by double immunostaining for CAV-1 and pathologische Anatomie Leiden endothelium (PAL-E, a PTC marker) in the PTCs of kidney allograft biopsy samples. The patients were divided into two groups: CAV-1/PAL-E<50% and CAV-1/PAL-E≥50%. Kaplan-Meier curves showed that CAV-1/PAL-E≥50% patients had a significantly worse prognosis than that of CAV-1/PAL-E<50% patients (log-rank; P<.001). C4d staining of PTCs was not associated with the development of graft failure (log-rank; P=.345), whereas in a multivariate Cox regression analysis, CAV-1 immunoreactivity in PTCs was independently associated with graft failure (hazard ratio: 11.1; P=.0324). CAV-1 immunoreactivity in PTCs may serve as a prognostic marker for kidney allograft survival.

AJKD Atlas of Renal Pathology: Acute T-Cell–Mediated Rejection

Acute cellular rejection, also called acute T-cell–mediated rejection (TCMR), presents in the transplant recipient with acute kidney injury and decreased urine output, and may be accompanied by proteinuria. In more severe cases there can be transplant tenderness. TCMR can develop at any time, as early as a week or as late as years after transplantation. With current immunosuppressive therapy, acute rejection develops in about 10%-12% of transplant patients. Pure TCMR responds well to increased immunosuppressive therapy. Light microscopy: Inflammation and tubulitis is scored. i1 is defined as inflammation involving 10%-25% of nonscarred parenchyma; i2 as 26%-50% involvement, and i3 as >50% involvement. Tubulitis is scored as t1 when 1-4 cells are present per tubular cross-section in non-atrophic tubules; t2 is 5-10 cells, and t3 is >10 cells. Diagnostic criteria for TCMR according to Banff classification 2005 (Solez et al, Am J Transplant 2007;7(3):518-526) include the following:•Type IA: significant interstitial inflammation (i2 or i3) and foci of moderate tubulitis (t2)•Type IB: significant interstitial inflammation (i2 or i3) and foci of severe tubulitis (t3)•Type IIA (mild vascular rejection): mild to moderate intimal arteritis, also called endothelialitis, involving <25% of the luminal circumference (v1)•Type IIB (moderate vascular rejection): severe intimal arteritis involving ≥25% of the luminal area (v2)•Type III (severe vascular rejection): transmural arteritis and/or arterial fibrinoid change and necrosis (v3)•Types II and III frequently also have antibody-mediated components Subtypes include plasma cell–rich cellular rejection and eosinophil-rich cellular rejection. However, the threshold for type I acute TCMR is high, and in clinical settings of presumed acute rejection, morphologic changes most often fall in the borderline category, defined as foci of tubulitis with minor interstitial inflammation (Banff i0 or i1), or interstitial inflammation (i2 or i3) with minor tubulitis (t1). Therefore, Banff working groups are assessing lower thresholds of i and t lesions for diagnosis of TCMR. The Cooperative Clinical Trials in Transplantation (CCTT) criteria set the threshold for acute TCMR as >5% of nonscarred parenchyma with inflammation, and at least 3 nonscarred tubules with tubulitis in 10 high-power fields. Biopsy adequacy for CCTT specificity was set as 2 pieces of cortex with 7 glomeruli and 2 arteries. Immunofluorescence microscopy: C4d is negative in peritubular capillaries in pure TCMR. Electron microscopy: No specific changes. Acute TCMR is due to cytotoxic T-cell reaction to HLA antigens on the donor kidney. These T cells can affect the tubules, interstitium, arteries, and even glomeruli. Mixed acute antibody-mediated rejection and TCMR can occur. Polyomavirus nephropathy causes interstitial inflammation and tubulitis, and is distinguished from type I TCMR by the pleomorphic infiltrate with lymphocytes, plasma cells, occasional polymorphonuclear neutrophils (PMNs) and eosinophils, viral cytopathic changes, and SV40 positivity by immunostaining. Acute tubulointerstitial nephritis due to drug hypersensitivity reaction may have more frequent eosinophils, but its appearance overlaps substantially with TCMR. Acute pyelonephritis has intratubular casts of PMNs and frequent PMNs in the interstitium. Acute antibody-mediated rejection may occur in combination with TCMR, and is diagnosed with positive serum donor-specific antibodies and positive C4d staining of peritubular capillaries and significant microcirculation inflammation (ie, inflammation in glomeruli and peritubular capillaries), and is suspected with type III changes described above. Endothelialitis in the transplant is highly indicative of type II TCMR, but recurrent vasculitis may produce a similar lesion, but usually with necrosis. Acute thrombotic microangiopathy may have occasional inflammatory cells or even fibrinoid necrosis. •Type I: Interstitial inflammation in unscarred cortex and tubulitis•Type II: Endothelialitis in artery/arterioleFigure 2Acute cellular rejection (T-cell mediated, type I) with interstitial lymphocytic infiltrate, foci of severe tubulitis, and interstitial edema (periodic acid–Schiff stain).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Acute cellular rejection (T-cell mediated, type I) with interstitial lymphocytic infiltrate, foci of mild tubulitis, and interstitial edema (Jones silver stain).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Acute cellular rejection (T-cell mediated, type II), also called acute vascular rejection, with interstitial lymphocytic infiltrate, foci of moderate tubulitis, and endothelialitis of an interlobular artery (Jones silver stain).View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Presentation and Outcomes of C4d-Negative Antibody-Mediated Rejection After Kidney Transplantation

The updated Banff classification allows for the diagnosis of antibody-mediated rejection (AMR) in the absence of peritubular capillary C4d staining. Our objective was to quantify allograft loss risk in patients with consistently C4d-negative AMR (n = 51) compared with C4d-positive AMR patients (n = 156) and matched control subjects without AMR. All first-year posttransplant biopsy results from January 2004 through June 2014 were reviewed and correlated with the presence of donor-specific antibody (DSA). C4d-negative AMR patients were not different from C4d-positive AMR patients on any baseline characteristics, including immunologic risk factors (panel reactive antibody, prior transplant, HLA mismatch, donor type, DSA class, and anti-HLA/ABO-incompatibility). C4d-positive AMR patients were significantly more likely to have a clinical presentation (85.3% vs. 54.9%, p < 0.001), and those patients presented substantially earlier posttransplantation (median 14 [interquartile range 8-32] days vs. 46 [interquartile range 20-191], p < 0.001) and were three times more common (7.8% vs 2.5%). One- and 2-year post-AMR-defining biopsy graft survival in C4d-negative AMR patients was 93.4% and 90.2% versus 86.8% and 82.6% in C4d-positive AMR patients, respectively (p = 0.4). C4d-negative AMR was associated with a 2.56-fold (95% confidence interval, 1.08-6.05, p = 0.033) increased risk of graft loss compared with AMR-free matched controls. No clinical characteristics were identified that reliably distinguished C4d-negative from C4d-positive AMR. However, both phenotypes are associated with increased graft loss and thus warrant consideration for intervention.

Development and validation of a prognostic index for allograft outcome in kidney recipients with transplant glomerulopathy

We studied 92 patients with transplant glomerulopathy to develop a prognostic index based on the risk factors for allograft failure within five years of diagnosis (Development cohort). During 60 months (median) follow up, 64 patients developed allograft failure. A chronic-inflammation score generated by combining Banff ci, ct and ti scores, serum creatinine and proteinuria at biopsy, were independent risk factors for allograft failure. Based on the Cox model, we developed a prognostic index and classified patients into risk groups. Compared to the low risk group (median allograft survival over 60 months from diagnosis), patients in the medium risk group had a hazard ratio of 2.83 (median survival 25 months), while those in the high risk group had a hazard ratio of 5.96 (median survival 3.7 months). We next evaluated the performance of the prognostic index in an independent external cohort of 47 patients with transplant glomerulopathy (Validation cohort). The hazard ratios were 2.18 (median survival 19 months) and 16.27 (median survival 1.6 months), respectively, for patients in the medium and high risk groups, compared to the low risk group (median survival 47 months). Our prognostic index model did well in measures of discrimination and calibration. Thus, risk stratification of transplant glomerulopathy based on our prognostic index may provide informative insight for both the patient and physician regarding prognosis and treatment.

AJKD Atlas of Renal Pathology: Chronic Antibody-Mediated Rejection

Chronic antibody-mediated rejection is commonly associated with progressive loss of kidney function and proteinuria, although early stages may be subclinical and recognizable only through protocol biopsies. Diagnostic criteria according to the Banff 2013 classification (Haas et al, Am J Transplant 2014:14(2):272-283) include the presence of all of the following:1.Histologic evidence of chronic tissue injury, defined by the presence of at least one of the following:a.Transplant glomerulopathy (cg > 0) in the absence of chronic thrombotic microangiopathyb.Severe peritubular capillary basement membrane multilayering by electron microscopyc.New-onset arterial intimal fibrosis with no other known etiology2.Current/recent histologic evidence of antibody interaction with vascular endothelium, defined by the presence of at least one of the following:a.Linear C4d staining in peritubular capillaries (at least 10% by immunofluorescence [IF] on frozen sections or any positivity by immunohistochemistry on paraffin sections)b.At least moderate microvascular inflammation (g + ptc > 2)c.Increased expression of tissue gene transcripts indicative of endothelial injury3.Presence of donor-specific antibodies (DSA; HLA or non-HLA) in the serum Similar to acute antibody-mediated rejection, endothelial injury results from interaction between DSA with primarily mismatched HLA or non-HLA molecules. Repetitive and chronic endothelial damage is associated with new basement membrane formation, which in glomeruli manifests as glomerular basement membrane duplication (transplant glomerulopathy), often with resulting segmental sclerosis, and multilayering of the peritubular capillary basement membrane. Podocyte injury and foot process effacement are common findings. Glomerular basement membrane duplication is also found in other conditions associated with chronic endothelial damage, causing a membranoproliferative pattern of injury. Chronic thrombotic microangiopathy may be indistinguishable from transplant glomerulopathy on a morphologic basis unless features of concomitant acute thrombotic microangiopathy or vascular changes are present. In such cases, correlation with clinical history will be important. Membranoproliferative glomerulonephritis due to immune complex processes (lupus nephritis, immunoglobulin A nephropathy, cryoglobulinemic glomerulonephritis, etc) must be ruled out by IF and electron microscopy. Peritubular capillary basement membrane multilayering, especially if not severe (<7 layers), is not specific to chronic antibody-mediated rejection. Arterial intimal fibrosis is a common finding, often associated with chronic hypertension and aging. Scattered T cells and foam cells may be present in the expanded intima, but are not specific for chronic antibody-mediated rejection and may also be seen in chronic T-cell–mediated rejection. •Transplant glomerulopathy•Peritubular capillary basement membrane multilayering•Peritubular capillary C4d stainingFigure 2Transplant glomerulopathy with prominent glomerular basement membrane duplication and cellular interposition in a capillary loop. Podocytes show significant foot process effacement (electron microscopy).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Chronic antibody-mediated rejection with severe peritubular capillary multilayering (>7 layers of basement membrane), representing response to repetitive and chronic endothelial injury. Note that the endothelial cell is swollen and has lost its normal fenestrations (electron microscopy).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Chronic antibody-mediated rejection with diffuse linear C4d staining in peritubular capillaries (immunofluorescence microscopy, C4d).View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Calcineurin Inhibitor Minimization With Ixazomib, an Investigational Proteasome Inhibitor, for the Prevention of Antibody Mediated Rejection in a Preclinical Model.

There is a need for new immunosuppression strategies to minimize calcineurin inhibitor (CNI) toxicity while effectively preventing antibody-mediated rejection (AMR). We tested the efficacy of an investigational proteasome inhibitor, ixazomib, alone and in a CNI minimization strategy in a rat kidney transplant model of transfusion-elicited acute AMR. Nonsensitized (naïve) and sensitized allograft recipients were randomized into 4 treatment groups (8 groups total, n = 3 to 6 in each group) and treated for 1 week. Groups included: no treatment, full dose cyclosporine (CsA, 10 mg/kg per day), ixazomib (0.25 mg/kg on days -5, -2 and +2) alone, and half dose CsA (5 mg/kg per day) + ixazomib. Compared to untreated animals, ixazomib alone or in combination with ½ dose CsA reduced donor-specific antibody, intragraft transcripts for chemokines CCL-21 and CXCL-13, and CD19 expression in both sensitized and naïve transplants. Compared to full dose CsA, the CNI minimization strategy with ixazomib inhibited AMR and allograft injury as evidenced by reduced C4d staining in peritubular capillaries, microcirculation inflammation, splenic plasma cells, circulating B cell activating factor, and intragraft transcripts for major histocompatibility complex class II, Toll-like receptors (TLR-1, TLR-10, and TLR-12) and CCL-21 and CXCL-13 in sensitized animals, indicating downregulation of B cell activation, antigen presentation and T-cell and B-cell signaling. These studies suggest that CNI minimization strategies including ixazomib are effective to prevent AMR including in sensitized kidney allograft recipients. Clinical studies are needed to determine the role of novel proteasome inhibitors for the prevention and treatment of AMR.

Clinical Implications of Angiotensin II Type 1 Receptor Antibodies in Antibody-mediated Rejection Without Detectable Donor-specific HLA Antibodies After Renal Transplantation

BackgroundSolid-phase immunoassays have improved detection sensitivity for donor-specific HLA antibody (DSHA) and permitted the accurate diagnosis of antibody-mediated rejection (AMR). However, DSHA is not always sufficient to explain the cause of AMR. Consequently, a means of assessing non-HLA antibodies is required to determine the cause of AMR. The aim of the present study was to evaluate the clinical implications of antibodies (Abs) targeting angiotensin II type I receptor (AT1R) in recipients with AMR but without serum DSHA. MethodsNon-HLA AMR cases diagnosed between January 2011 and June 2014 were included. Levels of anti-AT1R Abs (U/mL) were quantified by using AT1R assay kits (One Lambda, Calif, United States) with collected sera pretransplantation and at biopsy (cut-off value: 15 U/mL). ResultsSeventy-two patients were diagnosed with AMR during the above-mentioned period. Of them, 12 recipients (16.7%) had no DSHA. The sera of these 12 patients were tested (2 patients were only checked at time of biopsy). Nine patients (9/10) were presensitized for anti-AT1R Abs (median, 25.0 U/mL; range, 12.9 to 50.0 U/mL). Ten patients (10/12) were anti-AT1R− positive at time of biopsy (median, 23.2 U/mL; range, 11.4 to 50.0 U/mL). The mean time from transplantation to biopsy was 73 months. Eight patients experienced acute AMR, and 4 developed chronic AMR. Four patients showed negative C4d staining in peritubular capillaries (4/12). Patients were treated with plasmapheresis, low-dose intravenous immunoglobulin, and/or rituximab. ConclusionsAT1R Abs may play a significant role in AMR without detectable DSHA. Pretransplantation detection of AT1R Abs may be helpful for assessing the risk for non-HLA AMR.

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.

Late Kidney Dysfunction in a Kidney Transplant Recipient

Late kidney transplant dysfunction may be a harbinger of graft failure. For many years, calcineurin inhibitor toxicity was felt to be the main cause for graft dysfunction with fibrosis and transplant loss. Recently this idea has come into question. With the observation that peritubular capillary C4d staining in kidney allografts may indicate antibody-mediated injury in conjunction with biopsy study findings, an appreciation for antibody-mediated rejection as a major cause of late graft dysfunction and loss has emerged. Twenty percent to 30% of patients develop de novo donor-specific antibodies after kidney transplantation. There are no US Food and Drug Administration-approved treatments for antibody-mediated rejection, nor have any randomized controlled trials assessed efficacy. Off-label treatment strategies include some combination of plasma exchange, intravenous immunoglobulin, and rituximab. Other approaches, including splenectomy, bortezomib, and eculizumab, have also been tried.

Antibody-Mediated Rejection and Treatment in Pediatric Patients: One Center’s Experience

Antibody-mediated rejection is a rare complication that often results in the loss of the kidney graft. Treatment options include plasmapheresis, intravenous immunoglobulin, and use of rituximab. We retrospectively evaluated the data files from 86 pediatric renal transplant patients over the last 5 years. A biopsy was taken for each rejection episode. Seven patients (7.7%) developed antibody-mediated rejection. All patients with antibody-mediated rejection had histologic evidence of severe acute humoral rejection and extensive C4d staining in peritubular capillaries. Staining was diffuse (involving > 50% of peritubular capillaries) for 4 biopsies, and it was focal (involving < 50% of peritubular capillaries) for 3 biopsies. Twelve biopsies demonstrated at least 1 histologic feature associated with acute humoral rejection. Donor-specific antibodies were evaluated in recipients. The mean peak panel reactive antibody class 1 was 7.16% (range, 0%-86%). The mean time between rejection episodes and the transplant was 16.9 ± 13.5 months. All patients were treated with high-dose intravenous methylprednisolone and intravenous immunoglobulin. Three patients recovered renal function rapidly after this treatment. Donor-specific antibodies were negative in these patients. Five sessions of plasmapheresis were used simultaneously in these 4 patients. In 3 resistant patients, rituximab was prescribed after plasmapheresis and intravenous immunoglobulin. The presence of donor-specific antibodies was demonstrated in 4 patients. Two patients were refractory to antibody-mediated rejection treatment and lost their transplants. One patient had interstitial fibrosis and tubular atrophy during the 16th month after her antibody-mediated rejection. Graft survival in patients with antibody-mediated rejection at the end of 1 year was 71.4%. Early diagnosis and treatment with plasmapheresis, intravenous immunoglobulin, and rituximab may resolve antibody-mediated rejection. Although effective therapy is available for acute antibody-mediated rejection, the allograft remains at risk for chronic antibody-mediated rejection and shortened survival.

The specificity of acute and chronic microvascular alterations in renal allografts

The diagnosis of an antibody-mediated rejection (AMR) is made when there is evident histologic injury in the presence of detectable donor-specific alloantibodies (DSA) and diffuse peritubular capillary C4d staining (C4d-pos). In the presence of only detectable DSA or C4d-pos, the tissue injury is currently considered “presumptive” for antibody causation. In acute antibody-mediated rejection (AAMR), diagnostic morphologic features include microvascular inflammation (MVI), specifically glomerulitis and peritubular capillaritis. In the case of chronic active AMR (CAAMR), these inflammatory lesions have progressed to chronic microvascular injury, transplant glomerulopathy (TG) and peritubular capillary basement membrane multilayering (PTCBMML). Either TG or PTCBMML is sufficient morphological evidence for a diagnosis of CAAMR. Unfortunately, these lesions are not specific. MVI, TG, and PTCBMML are found in the setting of cell-mediated immunity, as well as in association with non-alloimmune mechanisms. The available treatments for AMR and CMR are different, and it is important to ascertain the dominant mechanism when approaching an individual patient. At present, no gold standard exists to establish the specific pathogenesis in the more ambiguous cases. We detail here the differential diagnosis of MVI, TG, and PTCBMML.

Renal Arteriolar C4d Deposition

The mechanism of kidney injury in hematopoietic stem cell transplantation (HSCT)-associated thrombotic microangiopathy (TA-TMA) is not completely understood. Renal C4d staining is a marker of classic complement activation and endothelial injury and has been described in preliminary reports of HSCT recipients with TA-TMA. Our objective was to evaluate complement in the pathogenesis of small vessel injury in children receiving HSCT. We hypothesized that kidney tissue from children with TA-TMA would more frequently show C4d deposition compared with HSCT recipients without histologic TA-TMA. We reviewed kidney specimens (biopsy or autopsy) from children who had undergone HSCT at a single center. Using histologic criteria alone, subjects were divided into TA-TMA (n = 8) and non-TA-TMA (control) groups (n = 12). C4d staining was performed by immunohistochemistry and evaluated on arterioles, peritubular capillaries, glomeruli, and tubular basement membranes. Diffuse or focal renal arteriolar C4d staining was more common in subjects with histologic TA-TMA (75%) compared with controls (8%). Rare peritubular capillary C4d staining was present in 50% of TA-TMA samples and was absent in controls. Glomerular C4d staining was seen at a similar frequency in cases and controls, whereas tubular basement membrane staining was less frequently observed and only in subjects with TA-TMA. Arteriolar C4d deposition may be a pathologic marker of TA-TMA, implicating localized complement fixation in HSCT recipients with kidney disease secondary to small vessel injury. Further studies to better characterize the preferential arteriolar C4d staining may identify a renal compartment of injury, possibly explaining the dramatic hypertension seen in TA-TMA.

Phospholipase A2 Receptor (PLA2R) Staining Is Useful in the Determination of De Novo Versus Recurrent Membranous Glomerulopathy

Membranous glomerulopathy (MG) is one of the most common glomerulonephritides involving the renal transplant. We sought to determine the utility of phospholipase A2 receptor (PLA2R) staining for the detection of recurrent MG. We also evaluated for increased evidence of antibody-mediated rejection in the de novo group, as some have reported. Twenty-two cases of MG occurring in renal transplant biopsies were identified, who had a tissue diagnosis documenting the primary native renal disease. There were 12 biopsies from 11 patients with recurrent MG and 12 biopsies from 11 patients with de novo MG. Morphologic evaluation and PLA2R staining was performed in all cases. Ten of 12 (83%) recurrent MG and 1 of 12 (8%) de novo MG biopsies showed positive glomerular staining for PLA2R, giving PLA2R a sensitivity of 83% (95% confidence interval, 51%-97%) and specificity of 92% (95% confidence interval, 60%-100%) for recurrent MG. There were 2 of 12 (17%) de novo and 1 of 12 (8%) recurrent biopsies showing the presence of microcirculation inflammation. Peritubular capillary C4d staining was negative in all cases. Recurrent MG is strongly correlated with PLA2R positivity, with a sensitivity of 83% and specificity of 92% for recurrent MG. There was no morphologic evidence of an association between antibody-mediated rejection and de novo MG, because both groups had a similar degree of microcirculation inflammation and peritubular capillary C4d staining. Most interestingly, PLA2R staining was almost always negative in de novo MG, suggesting a different mechanism in this unique form of MG.