Development Of Transplant Glomerulopathy Research Articles

P8.109: Simultaneous Vascular and Antibody-Mediated Rejection (AMR) in Pediatric Patients Caused Poor Prognosis in Renal Allograft

Introduction: The prognostic significance of simultaneous vascular rejection (VR) and AMR on graft survival is not clear enough, especially in the pediatric population. Accordingly, this study aims to determine the impact of simultaneous VR and AMR on the development of transplant glomerulopathy (TG) and interstitial fibrosis (IF) with the resulting graft survival. Methods: Forty-five recipients younger than 18 were separated into Group 1 patients (n:25) with pure AMR and Group 2 patients (n:20) with simultaneous VR+AMR. Tubular expression of TNF-α, TGF-β, and HLA-DR was evaluated. Peritubular capillary (PTC) and interstitial leukocytes were highlighted with TNF-α, HLA-DR, and CD68. The loss of HLA-DR expression on PTCs was studied to determine the PTC destruction. Diffuse IF and TG development were analyzed in the follow-up biopsies. Results: The response of Group 2 patients to rejection therapy was lower than Group 1 (p<0.001). PTC C4d expression was found higher in Group 2 than Group 1 (P<0.001). Group 2 showed a higher PTC destruction, IF, and TG incidence than Group 1(P<0.01). The development of IF and TG increases with the increasing degree of glomerulitis, C4d expression, and PTC destruction (p<0.01). Tubular and interstitial TNF-α, TGF-β, and HLA-DR expressions were found higher in Group 2 compared to Group 1 (p<0.01). The degree of PTC destruction and C4d expression increased with increasing leukocyte and macrophage infiltration in PTCs and interstitium (p<0.001). The development time of IF and TG decreased with increasing intensity of PTC and interstitial infiltration, glomerulitis, PTC destruction, and C4d expression (p<0.01). Also, the development of IF and TG shortened with increasing HLA-DR, TNF-α expression in inflammatory cells and increasing TNF-α, TGF-β, HLA-DR expression in tubular cells (P<0.01). Overall, the 1-, 3- and 5-year graft survival was 96%, 92%, and 79%, respectively, for Group 1 patients, while 95%, 40%, and 10% respectively for Group 2 recipients (p<0.001). Conclusion: The prognosis and course of antibody-mediated vascular rejection are noticeably different from pure AMR, with antibody-mediated vascular rejection having the poorest outcome through leading the early development of IF and TG via augmenting inflammatory and fibrotic pathways. Thus, developing new treatment strategies for antibody-mediated vascular rejection could salvage many kidney allografts.

Rearrangement of the Endothelial Cell Cytoskeleton in Glomerular and Peritubular Capillaries after Antibody-Mediated Rejection: Significance in the Development of Transplant Glomerulopathy and Interstitial Fibrosis

Rearrangement of the Endothelial Cell Cytoskeleton in Glomerular and Peritubular Capillaries after Antibody-Mediated Rejection: Significance in the Development of Transplant Glomerulopathy and Interstitial Fibrosis

Immunoglobulin isotype switching of antibodies to vimentin is associated with development of transplant glomerulopathy following human renal transplantation

BackgroundImmune responses to tissue-restricted self-antigens are thought to play a role in chronic rejection after solid organ transplantation. De novo development of antibodies (Abs) to vimentin have been reported to be associated with interstitial fibrosis/tubular atrophy after kidney transplant, and it has been suggested that immunoglobulin isotype switching of Abs to vimentin may occur during this process. We aimed to determine the correlation between immunoglobulin isotype switching of Abs to vimentin and development of transplant glomerulopathy (TG) after kidney transplant, and to determine whether citrullinated modification of vimentin is required for de novo anti-vimentin development. MethodsSera were collected from 24 patients with TG (diagnosed on biopsy), 24 matched stable kidney transplant recipients (KTxRs) and 22 normal healthy subjects who did not undergo transplant. Serum vimentin Abs concentrations were measured using enzyme-linked immunosorbent assay (ELISA). Immunoglobulin isotypes of anti-vimentin were determined using isotype-specific Abs conjugated with horseradish peroxidase. Samples were considered positive to vimentin Abs if the values were above mean+2× standard deviations of the levels in the healthy control subjects. Specificities of anti-vimentin for mutated citrullinated vimentin and anti-mutated citrullinated vimentin were measured by ELISA. ResultsIn this retrospective analysis of 24 KTxRs with TG, 16/24 (67%) patients with biopsy-proven TG developed Abs to vimentin (645±427ng/ml). In contrast, only 4/24 (17%) stable KTxRs had detectable Abs to vimentin (275±293ng/ml; p=0.001). Of the patients with TG, 15/24 (63%) developed Abs to vimentin of IgG isotype (572±276ng/ml), whereas only 6/24 (25%) stable KTxRs (310±288ng/ml) had anti-vimentin of IgG isotype (p=0.002). However, no significant difference was noted in the concentration of IgM isotype anti-vimentin between KTxRs with TG (9/24 [38%], 407±401ng/ml) and stable KTxRs (5/24 [21%], 348±439ng/ml; p=0.631). The serum concentration of Abs specific for the mutated form of citrullinated vimentin was not significantly different between KTxRs with TG and stable KTxRs. ConclusionsPatients with biopsy-proven TG demonstrated significantly increased levels of anti-vimentin Abs of the IgG isotype compared with stable KTxRs. Anti-vimentin in stable KTxRs was primarily of IgM isotype. Therefore, the observed isotype switching of anti-vimentin from IgM to IgG isotype strongly suggests ongoing immune responses to vimentin in KTxRs diagnosed with TG. Furthermore, as opposed to patients with rheumatoid arthritis (who develop immune responses primarily to citrullinated vimentin), KTxRs diagnosed with TG developed immune responses to non-citrullinated vimentin, suggesting that modification of vimentin protein via citrullination is not required for the de novo anti-vimentin response seen in patients with TG.

Development of Transplant Glomerulopathy in Recipients With Antibody Mediated Rejection

Introduction: Activation of PARP is a critical factor in the pathogenesis of various inflammatory and fibrotic process. Thus, we aimed to show the integral of PARP in the inflammatory process of glomeruli and the development of transplant glomerulopathy (TG). Methods: Biopsies of 82 patients with antibody-mediated rejection were included to study. PARP, α-SMA, and TNF-α, expression of glomeruli studied. The mean number of neutrophil, macrophage, and lymphocyte infiltration per glomerulus evaluated. The degree of the peritubular capillary (PTC) inflammation and the presence of neutrophil and macrophage in PTCs investigated. PTC C4d, PARP, and HLA-DR expression graded. The decreasing intensity of PTC HLA-DR (PTC-DR) expression accepted as the increasing degree of the destruction of PTC’s. Follow-up biopsies analyzed for the development of transplant glomerulopathy. Results: Of 82 recipients, only 40 showed TG. The time of the development of TG decreased with increasing degree of PTC neutrophil and macrophage infiltration, PTC inflammation, intraglomerular macrophage, and neutrophil infiltration (p < 0.001). TG development is increased with increasing amount of C4d expression and with decreasing degree of PTC-DR expression (p < 0.001). TG was found earlier in recipients who had a higher degree of glomerular, and PTC PARP expression (p < 0.001). Also, patients with a greater degree of glomerular PARP expression showed a higher amount of glomerular α-SMA and TNF-α expression. The intensity of the glomerular and PTC macrophage and neutrophil infiltration increases with increasing amount of glomerular and PTC PARP expression (p < 0.001). The 5-year graft survival was 78% and 15% for recipients with negative and positive glomerular PARP expression respectively (p < 0.001). Also, the5-year survival was 84%, 41% and %0 for patients with grade1, 2 and 3 PTC PARP expression respectively (p < 0.001). Conclusion: Increased PARP activation both in glomeruli and PTCs leads to early TG and early graft loss. Thus, it will be beneficial to use PARP inhibitor drugs in recipients with a high risk of transplant glomerulopathy.

Early subclinical inflammation correlates with outcomes in positive crossmatch kidney allografts.

The aim of this study was to investigate correlations between early subclinical findings (10- and 90-day histology and gene expression data) and late outcomes (transplant glomerulopathy and graft loss) in positive crossmatch kidney transplants (+XMKTx). We compared 34 +XMKTx (19 receiving eculizumab and 15 receiving standard of care without eculizumab) to 13 -XMKTx (between August 2001 and August 2011). At 10days, light microscopy identified subclinical inflammation in only 18% of +XMKTx, while intragraft gene expression identified inflammation in 79% (gene sets for activated macrophages, dendritic cells, NK cells or T cells). Inflammation persisted at 90days and was associated with the development of transplant glomerulopathy by 2years and graft loss. In contrast, endothelial cell (EC) changes present at 90days by either electron microscopy or gene expression were not associated with transplant glomerulopathy or graft loss in this cohort. Eculizumab treatment did not appear to alter inflammation or EC changes. Therefore, intragraft inflammation might be an appropriate surrogate marker of progression and also a target of therapy to prevent chronic antibody-mediated rejection.

Benefits of rituximab combined with intravenous immunoglobulin for desensitization in kidney transplant recipients.

Highly HLA-sensitized (HS) patients have difficulty accessing compatible donors, especially deceased donor (DD) transplants. Desensitization protocols (DES) have evolved, but rigorous evaluation is lacking. Here, we examined the efficacy of rituximab as a DES agent in a placebo-controlled trial. Candidates were randomized to IVIG+placebo versus IVIG+rituximab. End points included rates of transplantation, antibody-mediated rejection (ABMR), and renal function. Protocol biopsies were performed at 1 year and analysis of patient and graft survival and donor-specific HLA antibodies (DSA) were performed. Initially, 15 HS DDs were randomized with 13 receiving transplants. However, we discontinued study entry after five serious adverse events were observed. The study was un-blinded and attribution of patients was noted (IVIG+placebo N=7, IVIG+rituximab N=6). No significant differences were seen in DSA levels at transplant. All ABMR episodes occurred in the IVIG+placebo arm and required intense therapy (P=0.06). The two graft losses were in the placebo group. DSA rebound associated with severe ABMR was seen in three patients in the IVIG+placebo group. No rebound was seen in the IVIG+rituximab group. Renal function at 6 and 12 months showed a significant benefit for IVIG+rituximab (P=0.04). Based on limited assessment with acknowledged limitations, both protocols appear effective in achieving levels of DSA allowable for transplantation. However, IVIG+rituximab appeared more effective in preventing DSA rebound and, more importantly, preventing ABMR and development of transplant glomerulopathy.

Acute Cellular Rejection

Mixed rejection in kidney transplantation consists of histologic and/or serological evidence of both cellular and humoral components. As it is not confined to a distinct category in the Banff classification, how to best manage these patients is not clear. The aim of this study was to determine the incidence and outcome of morphological T-cell-mediated rejection (TCMR) with a humoral component, defined as the presence of either DSA or C4d, compared with the outcome of pure TCMR. We retrospectively studied 922 consecutive renal transplant recipients and analyzed patients with TCMR according to the evidence of a humoral component. A total of 147 cases of morphological TCMR were analyzed. Of these, 92 (62.6%) had "pure" TCMR and 55 (37.4%) had "mixed" TCMR on the index biopsy. On univariant analysis, diffuse C4d (odds ratio [OR]=10.9, 95% confidence interval [CI]=1.8-66.9, P=0.01) and DSA positivity at the time of index biopsy (OR=2.8, 95% CI=1.2-6.6, P=0.02) were associated with allograft loss, whereas arteritis (OR=0.5, 95% CI=0.2-1.2, P=0.11) and glomerulitis (OR=0.9, 95% CI=0.4-2.1, P=0.8) were not. Arteritis was associated with subsequent antibody-mediated rejection (OR=4.9, 95% CI=1.1-20.8, P=0.03), and glomerulitis was associated with the development of transplant glomerulopathy (OR=10.7, 95% CI=3.1-37.1, P<0.01). On the multivariate analysis, only patients with C4d and DSA were at risk of graft failure (OR=4.9, 95% CI=2.0-12.0, P<0.01) in the medium term. TCMR with a humoral component has a worse prognosis when compared with pure TCMR. As such, it is important to test for alloantibody in cases of morphological TCMR to optimize patient management. Such cases might benefit from more aggressive immunotherapy.

Pathogenesis of Transplant Glomerulopathy

Transplant glomerulopathy is a late complication of renal transplantation. The characteristic morphology of transplant glomerulopathy includes thickening of glomerular capillary loops with double contour, and duplication of glomerular basement membrane on electron microscopy. Clinical and experimental evidences support the role of antibody-mediated immune mechanism in the development of transplant glomerulopathy. Antibody-induced endothelial cell injury is the key pathogenesis of transplant glomerulopathy. The evolution of transplant glomerulopathy in the context of immunologic injury is briefly reviewed.

Antibodies Reactive to Non-HLA Antigens in Transplant Glomerulopathy

Although T and B cell alloimmunity contribute to transplant injury, autoimmunity directed at kidney-expressed, non-HLA antigens may also participate. Because the specificity, prevalence, and importance of antibodies to non-HLA antigens in late allograft injury are poorly characterized, we used a protein microarray to compare antibody repertoires in pre- and post-transplant sera from several cohorts of patients with and without transplant glomerulopathy. Transplantation routinely induced changes in antibody repertoires, but we did not identify any de novo non-HLA antibodies common to patients with transplant glomerulopathy. The screening studies identified three reactivities present before transplantation that persisted after transplant and strongly associated with transplant glomerulopathy. ELISA confirmed that reactivity against peroxisomal-trans-2-enoyl-coA-reductase strongly associated with the development of transplant glomerulopathy in independent validation sets. In addition to providing insight into effects of transplantation on non-HLA antibody repertoires, these results suggest that pretransplant serum antibodies to peroxisomal-trans-2-enoyl-coA-reductase may predict prognosis in kidney transplantation.

A Critical Appraisal of Methods to Grade Transplant Glomerulitis in Renal Allograft Biopsies

Transplant glomerulitis is an increasingly recognized lesion in renal transplant biopsies. To develop a refined grading system, we defined glomerulitis by the presence of ≥5 leukocytes/glomerulus and evaluated 111 biopsies using three different grading systems: (i) percentage of glomerular involvement, (ii) peak inflammation in the most severely affected glomerulus and (iii) presence/absence of endocapillary occlusion by inflammatory cells. Endocapillary occlusion had no impact on graft survival, but was associated with increased serum creatinine, proteinuria and subsequent transplant glomerulopathy. Grading based on either percent or peak glomerular involvement correlated with graft failure and peritubular capillaritis. However, the percent glomerular involvement method had the additional advantage of displaying associations with: concurrent proteinuria, focal or diffuse immunoperoxidase peritubular capillary C4d staining, 1-year postbiopsy serum creatinine, subsequent detection of donor-specific antibody and development of transplant glomerulopathy. Patients with >75% glomerular involvement also revealed persistent high-grade glomerulitis on follow-up biopsies despite antirejection treatment. In conclusion, grading of glomerulitis is a meaningful exercise, and a quantification system based on percentage of glomerular involvement shows the most robust associations with clinical parameters and prognosis.

Clinical significance of anti-HLA antibodies detected by Luminex ®: Enhancing the interpretation of CDC-BXM and important post-transplantation monitoring tools

B-cell crossmatch (BXM) was originally introduced to increase the sensitivity to detect anti-HLA antibodies of conventional CDC crossmatch in renal transplantation. Newer techniques such as Luminex ® have greater sensitivity in detecting anti-HLA antibodies but have not been directly evaluated versus BXM. We discuss our experience with Luminex testing and the significance of donor-specific antibodies (DSA) defined by Luminex in three populations, as compared with the CDC crossmatch. In the general transplant population, Luminex-defined DSA were found in only one third of positive CDC-BXM and were associated with graft rejection. Luminex testing enhanced the interpretation of CDC-BXM and identified patients with clinically relevant BXM. In the highly sensitized transplant population, Luminex-defined DSA were found in two thirds of positive BXM and were a better predictor of graft rejection. Therefore, Luminex assays rather than CDC-BXM should be used to facilitate kidney allocation in highly sensitized patients. In the post-transplantation population, Luminex antibody monitoring for DSA was shown to be important, as it defined low-level de novo DSA that were associated with development of transplant glomerulopathy and a significant predictor of graft loss in those patients. Thus Luminex testing facilitated the interpretation of CDC-BXM and provided a useful predictive tool for the detection of clinically significant DSA in post-transplantation antibody monitoring.

Transplant Glomerulopathy May Occur in the Absence of Donor-Specific Antibody and C4d Staining

Transplant glomerulopathy (TGP) has been proposed to be a component of chronic antibody-mediated rejection (AMR). We have studied 36 patients with TGP and 51 patients with chronic allograft nephropathy (CAN) but without TGP for C4d staining and donor-specific anti-HLA antibodies (DSA) to investigate the alloantibody-mediated mechanisms. Allograft biopsies were stained with C4d staining and DSAs were studied by Luminex Flow Beads. Allograft biopsies were done at a mean of 5.3 +/- 5.0 and 5.6 +/- 4.6 yr after transplantation in patients with CAN and TGP, respectively. The mean creatinine level at the time of the biopsy was 2.7 +/- 1.2 mg/dl in each group. Proteinuria of >1.0 g/d was more common in patients with TGP (61 versus 25%; P = 0.002). Whereas three patients with TGP had a history of acute AMR, none of the patients with CAN had. Mean chronicity score of the biopsies were 1.7 +/- 0.7 in patients with CAN and 1.9 +/- 0.8 in patients with TGP. Biopsies from only two (4%) patients with CAN and four (11%) patients with TGP had diffuse C4d positivity. DSA were found in 36% of TGP and 33% of CAN patients. These results suggest that a substantial number of patients with TGP did not have positive C4d staining or DSA, indicating that a non-alloantibody-mediated process may be involved in the development of TGP in some patients.

Impact of HLA Antibodies on Transplant Glomerulopathy

The influence of humoral rejection on the development of chronic allograft nephropathy (CAN) is controversial, especially in relation to transplant glomerulopathy. The aim of our study was to analyse the influence of anti-HLA antibodies on the development of transplant glomerulopathy ( cg0, cg1, cg2, and cg3; Banff’97). We selected all renal transplants patients from 1975 to 2003 who had a functioning graft for at least 6 months and a clinically indicated graft biopsy with CAN and chronic glomerular changes (case group). We studied the presence of anti-HLA antibodies (Ab) in the last serum taken while the graft was functioning and divided them into three groups according to the severity of glomerular lesions. We also selected 52 contemporary and comparable cases without transplant glomerulopathy (control group). A total of 77 case had transplant glomerulopathy: 39 cg1, 29 cg2, and 9 cg3. Pretransplant Ab titers and number of previous blood transfusions were higher among the subgroup with the most severe glomerulopathy. Patients who developed posttransplant anti-HLA Ab more frequently showed transplant glomerulopathy. Serum creatinine and proteinuria were higher among cases with chronic glomerulopathy, and more grafts were lost in that group. Thus, the presence of HLA-Ab is a key factor in the development of transplant glomerulopathy and chronic allograft rejection.

Histopathology of chronic renal allograft dysfunction

The rate of late allograft loss remains relatively constant, despite greatly improved success in the early management of renal transplants. The pathological changes encountered in kidneys undergoing late allograft dysfunction are the result of a variety of processes, both immune and nonimmune. The gradual appearance of interstitial fibrosis and tubular atrophy is a nonspecific finding, which characterizes late allograft dysfunction. Features that indicate chronic rejection include transplant glomerulopathy, vascular intimal hyperplasia, particularly in association with intimal lymphocytic infiltration. Both of these changes may be related to humoral rejection. Animal models provide important insights into the mechanism of chronic rejection. In a commonly used model, the Fisher to Lewis rat model, antidonor antibodies against matrix proteins are associated with the development of transplant glomerulopathy, and the appearance of antitubular antibodies and a granulomatous interstitial nephritis may indicate graft-host differences in tubular basement membrane structure.

EFFECT OF PROTEIN A IMMUNOADSORPTION ON PANEL LYMPHOCYTE REACTIVITY IN HYPERIMMUNIZED PATIENTS AWAITING A KIDNEY GRAFT

Six hyperimmunized patients awaiting a kidney graft underwent immunoadsorption of separated plasma through a protein-A column to remove HLA antibodies. This procedure was partially limited by constant and rapid antibody resynthesis in spite of strong immunosuppression with cyclophosphamide and prednisolone. Only two patients could be grafted with previously positive--currently negative crossmatches. The first died of infection on day 40, having developed early chronic vascular rejection and the other returned to hemodialysis on day 285 after the development of transplant glomerulopathy. However, immunoadsorption seems to be effective in removing HLA antibodies having a titer below 1/2. Such extremely hyperimmunized patients should probably be excluded from the immunoadsorption program.