Antibody-mediated Renal Rejection Research Articles

Photopheresis Abates the Anti-HLA Antibody Titer and Renal Failure Progression in Chronic Antibody-Mediated Rejection.

Chronic renal antibody-mediated rejection (ABMR) is a common cause of allograft failure, but an effective therapy is not available. Extracorporeal photopheresis (ECP) has been proven successful in chronic lung and heart rejection, and graft versus host disease. The aim of this study was to evaluate the effectiveness of ECP in chronic ABMR patients. We investigated ECP treatment in 14 patients with biopsy-proven chronic ABMR and stage 2-3 chronic renal failure. The primary aim was to e valuate the eGFR lowering after 1 year of ECP therapy. The ECP responders (R) showed eGFR reduction greater than 20% vs the basal levels. We also evaluated the effectiveness of ECP on proteinuria, anti-HLA antibodies (HLAab), interleukin 6 (IL-6) serum levels, and CD3, CD4, CD8, CD19, NK, Treg and T helper 17 (Th17) circulating cells. Three patients dropped out of the study. The R patients were eight (72.7%) out of the 11 remaining patients. Because ECP was not associated with any adverse reaction, the R patients continued such treatment for up to 3 years, showing a persisting eGFR stabilization. Twenty four hour proteinuria did not increase in the R patients over the follow-up when compared to the non-responder patients (NR). In the R patients, the HLAab levels were reduced and completely cleared in six out of eight patients when compared with the NR patients. The NR HLAab levels also increased after the discontinuation of the ECP. The ECP in the R patients showed a decrease in CD3, CD4, CD8, CD19, and NK circulating cells. The ECP treatment in the R patients also induced Tregs and Th17 cell increases, and a decrease of the IL-6 serum levels. ECP abates the HLAab titer and renal failure progression in patients with chronic renal ABMR, modulating the immune cellular and humoral responses.

Successful Treatment of Severe COVID-19 Pneumonia With Clazakizumab in a Heart Transplant Recipient: A Case Report

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is characterized by an overwhelming cytokine response. Various treatment strategies have been attempted. Methods and ResultsA 61-year-old man with heart transplantation in 2017 presented with fever, cough, and dyspnea, and was confirmed positive for coronavirus disease 2019 (COVID-19). Laboratory tests showed significant elevations in C-reactive protein and interleukin-6 (IL-6). Echocardiogram showed left ventricular ejection fraction 58% (with ejection fraction 57% 6 months prior). Given the lack of clear management guidelines, the patient was initially managed symptomatically. However, the patient subsequently had a rapid respiratory deterioration with worsening inflammatory markers on day 5 of admission. Tocilizumab (anti-IL-6R) was in low supply in the hospital. The patient was offered clazakizumab (anti-IL-6) for compassionate use. Patient received 25 mg intravenously × 1 dose. Within 24 hours, he showed significant improvement in symptoms, oxygen requirements, radiological findings, and inflammatory markers. There was a transient leukopenia that improved in 4 days. He was discharged home on day 11, with negative nasopharyngeal SARS-CoV-2 PCR as an outpatient on day 35, development of positive serum COVID-19 IgG antibody, and he continued to do well on day 60, with no heart-related symptoms. ConclusionClazakizumab is a monoclonal antibody against human IL-6, which may be helpful in inhibiting the cytokine response to SARS-CoV-2 in COVID-19. Although not yet FDA approved, it is being investigated for treatment of renal antibody-mediated rejection. Clinical trials of clazakizumab for treatment of COVID-19 are underway worldwide.

TO007PLASMA EXTRACELLULAR VESICLES MEDIATE ENDOTHELIAL TO MESENCHYMAL TRANSITION AND TUBULAR SENESCENCE IN RENAL ANTIBODY MEDIATED REJECTION BY COMPLEMENT ACTIVATION

Abstract Background and Aims EVs (Extracellular vesicles) are circulating microparticles able to mediate cell-to cell communication by carrying proteins, DNA, RNA or antibodies on their surface. EV are emerging as pivotal in renal Antibody-mediated rejection (AMR), one of the major causes of transplant failure associated to a massive complement activation. AMR is characterized by endothelial to mesenchymal transition (EndMT) and the occurrence of tubular accelerated senescence process, known as graft “Inflammaging”. However, the potential role of EVs in accelerating the renal aging and graft fibrosis after AMR is not well understood. Method Plasma EVs were isolated from 10 Acute AMR (AAMR), 10 Chronic AMR (CAMR) patients, 5 stable graft transplanted patients and 5 healthy volunteers. EVs were isolated by ultracentrifugation at 100.000 g for 1h at 4°C, quantified by Nanoparticle Tracking Analysis (Nanosight, NTA EV/ml) and characterized by FACS (Attune Nxt). RPTEC and endothelial cell culture were incubated with EVs (5e+4 EVs/cells target for 24h. MTT test was performed to assess cell viability. Cellular senescence was investigated by qPCR for p21, p53, Klotho and CYP1B1 and SA-β-gal staining were performed. To assess EndMT analysis for CD31, VE-cadherin, Vimentin, Collagen I was performed by FACS. mRNA level of C3 and CFH were also measured by qPCR and C4d deposits were evaluated in endothelial cell colture by IF. Renal biopsies were then analyzed for inflammaging (p16INK4a) and EndMT markers (CD31/αSMA) by IHC and IF. Results The Nanosight analysis showed significant differences in the amount of-EVs count per ml of plasma in AMR patients compared to healthy subjects. EVs appeared to be significantly augmented in acute AMR, in a higher manner than chronic AMR (NTA, p=0.0154). By cytofluorimetric analysis, the endothelial markers CD31 and VE- cadherin appeared to be significantly increased compared of healthy control, indicating a predominant endothelial EV origin (p<0.05). Furthermore, an increase for CD3, CD4, CD8, CD40 and CD40L lymphocyte/monocytes markers was found. In vitro, the exposure of RPTEC and endothelial cells to AMR-derived EVs did not induce the loss of cell proliferation. However, AMR-derived EVs induced senescence in RPTEC, as observed by increase in SA-β-gal positive cells, upregulation of p21, p53, CYP1B1 and downregulated KL gene expression (p<0.05). EVs induced the EndMT as observed by FACS with the downregulation of CD31, VE-cadherin (CD144) and increase of Vimentin and Collagen I (p=0.025). To evaluate the contribution of EVs to local complement activation, C3 and CFH qPCR analysis were performed. EVs from AMR patients induced a significant increase in C3 gene expression with concomitant downregulation in CFH in RPTEC. EV exposition induced the classical and lectin pathway activation in endothelial culture medium, and C4d deposition. These data support the hypothesis that circulating EVs can amplify local complement activation in systemic endothelial and tubular cells during AMR, therefore leading to accelerated senescence and fibrosis as later effects. Finally, renal AMR biopsies showed significant tubular senescence as indicated by p16 expression; p16 was significantly upregulated in Chronic compared with Acute AMR biopsies (p<0.05). The AMR biopsies showed positivity for EndMT, as indicated by CD31 decrease and interstitial αSMA upregulation (p<0.05). Conclusion These results suggest a putative role for circulating AMR derived-EVs in inducing the tubular inflammaging by local complement activation and early fibrosis by EndMT. The EVs cargo characterization that is ongoing might highlight novel targets for therapeutic intervention.

Outcomes of Highly Sensitized Patients Undergoing Simultaneous Liver and Kidney Transplantation: A Single-Center Experience With Desensitization

BackgroundPreformed donor-specific human leukocyte antigen antibodies (DSAs) in patients undergoing simultaneous liver and kidney transplantation (SLKT) are an independent risk factor for poorer patient and renal allograft survival. The outcomes of patients highly sensitized (HS) against HLA antigens undergoing SLKT and select HS SLKT recipients undergoing desensitization at a high-volume desensitization center were investigated. MethodsSeventy-five patients undergoing SLKT at a high-volume desensitization center between January 1, 2001, and December 31, 2015, were retrospectively reviewed. HS patients were defined by panel-reactive antibody (PRA) >30% (n = 17 patients), 11 of whom received pre- or perioperative desensitization with high-dose intravenous immunoglobulin (IVIG) ± rituximab. ResultsHS patients had significantly higher class I and class II PRA (class I = 41.3% ± 40.0% vs 2.5% ± 6.3%; class II = 45.7% ± 36.4% vs 1.0% ± 2.9%; P < .001), were more likely to be female (P = .05), and more likely to have had a prior transplant (P = .03). HS patients demonstrated greater susceptibility to renal cell-mediated rejection (CMR) (23.5% vs 5.2%, P = .02) compared to nonsensitized patients. Higher renal antibody-mediated rejection (ABMR) was also observed in HS patients, 11.8% vs 3.4%, but did not reach significance (P = .18). Desensitization in select HS SLKT patients was well tolerated but did not improve patient and allograft survival or significantly curtail rejection. ConclusionHS SLKT recipients demonstrated increased allograft rejection, particularly CMR, but patient and graft survival were not impacted in the first year post-transplant. Select HS SLKT patients tolerated desensitization with high-dose IVIG ± rituximab and may have received additional immunoprotection against ABMR but survival was not affected.

Memory CD4 T Cells Induce Antibody-Mediated Rejection of Renal Allografts.

Despite advances in immunosuppression, antibody-mediated rejection is a serious threat to allograft survival. Alloreactive memory helper T cells can induce potent alloantibody responses and often associate with poor graft outcome. Nevertheless, the ability of memory T cells to elicit well characterized manifestations of antibody-mediated rejection has not been tested. We investigated helper functions of memory CD4 T cells in a mouse model of renal transplantation. Whereas the majority of unsensitized C57Bl/6 recipients spontaneously accepted fully MHC-mismatched A/J renal allografts, recipients containing donor-reactive memory CD4 T cells rapidly lost allograft function. Increased serum creatinine levels, high serum titers of donor-specific alloantibody, minimal T cell infiltration, and intense C4d deposition in the grafts of sensitized recipients fulfilled all diagnostic criteria for acute renal antibody-mediated rejection in humans. IFNγ neutralization did not prevent the renal allograft rejection induced by memory helper T cells, and CD8 T cell depletion at the time of transplantation or depletion of both CD4 and CD8 T cells also did not prevent the renal allograft rejection induced by memory helper T cells starting at day 4 after transplantation. However, B cell depletion inhibited alloantibody generation and significantly extended allograft survival, indicating that donor-specific alloantibodies (not T cells) were the critical effector mechanism of renal allograft rejection induced by memory CD4 T cells. Our studies provide direct evidence that recipient T cell sensitization may result in antibody-mediated rejection of renal allografts and introduce a physiologically relevant animal model with which to investigate mechanisms of antibody-mediated rejection and novel therapeutic approaches for its prevention and treatment.

68: Predictive markers for the detection of antibody-mediated rejection in kidney transplantation : Procalcitonin and CH50

Aims Antibody-mediated rejection (AMR) is the leading cause of early allograft loss, chronic rejection and graft failure. The aim of this research is to clarify useful predictive serum marker for occurrence of AMR in patients after kidney transplantation (KT). Methods Forty-two KT patients were studied; 32 with AMR and 10 without AMR. The AMR is confirmed by presence of donor-specific antibodies and positive C4d staining on renal biopsy. Plasma levels of IL-6, procalcitonin (PCT) and CH50 were measured in KT patients and 40 healthy subjects. Statistical analyses were performed by Kruskal–Wallis test. Results Median levels of CH50 were 18.20, 46.40 and 28.15 U/mL in KT patients with AMR, KT patients without AMR, and healthy subjects, respectively. And, PCT levels were 0.045, 0.033 and less than 0.020 ng/mL; and IL-6 were 1.93, 3.48, and less than 1.50 pg/mL, respectively. IL-6 and PCT showed no statistically significant differences, but CH50 was significantly decreased in the group with AMR than without AMR after KT ( p p = 0.0020) and without AMR (0.0335 ng/mL, p = 0.0010). Conclusions The significant decline of CH50 in KT patients with AMR may implicate that the complement system participates in renal antibody-mediated rejection and CH50 would be useful predictive marker for the detection of AMR in KT patients. Also, further evaluation on the usefulness of PCT is needed as a predictive marker of early AMR in KT.

Proteasome inhibitors in progressive renal diseases

Proteasome (PS) is a sophisticated protein degradation machinery comprising a 20S proteolytic core particle provided with caspase-like, trypsin-like and chymotrypsin-like activities on ubiquitinilated proteins. The products of this selective, complex, controlled and strictly coordinated system play a crucial role in cell cycle progression and apoptosis; activation of transcription factors, cytokines and chemokines; degradation and generation of MHC class I-presented peptides. PS has recently emerged as a promising drug target in cancer therapy, and bortezomib has been approved for refractory multiple myeloma. PS proteolysis is crucial for the degradation of the inhibitory protein IkB of nuclear factor kB (NF-kB), and hence, an interesting field of research has been developed on possible benefits of drugs with anti-PS activity in disease conditions with hyper-expression of NF-kB. PS inhibitors are being adopted in pilot studies in antibody-mediated renal rejection and in AL amyloidosis, with increasing scientific interest in possible applications in lupus, IgA nephropathy, idiopathic nephrotic syndrome and renal fibrosis. The most often used PS inhibitor, bortezomib, has a severe peripheral neurotoxicity, and the search for effective and less toxic PS-targeted drugs is a challenging area also in nephrology.

Impact of donor-specific antibodies on results of liver transplantation

To critically examine the recent literature evaluating the importance of HLA donor-specific antibody (DSA) impact on liver transplant and simultaneous liver-kidney transplant (SLKT) outcomes. Many preformed DSAs, especially of low mean fluorescence intensity (MFI), are absorbed by the liver at transplant. However, patients with post-liver transplant DSA, especially of higher MFI, are at increased risk of acute and chronic rejection. C4d staining, when positive, may be helpful but lacks sensitivity especially in formalin tissue. SLKT recipients may need close follow-up when class II DSA is found, as the liver protects the kidney from hyperacute rejection, but can still cause early renal antibody-mediated rejection, liver allograft rejection, and impair patient, liver allograft, and renal allograft survival. Some DSAs are relevant in liver transplant and can lead to acute and chronic allograft rejection. However, before clinical practice patterns can change we must create unified diagnostic criteria, define the pathologic potential of different DSAs, and improve the specificity of current testing.

Peritubular capillary C4d deposition in lupus nephritis different from antibody-mediated renal rejection

C4d deposition in peritubular capillaries (PTC) has been used as a marker of antibody-mediated rejection (AMR). However, PTC C4d deposition is not described in patients with lupus nephritis (LN). C4d deposition in PTC was detected in 455 patients with biopsy proven LN in the present study. Renal tissues from 21 cases of acute AMR served as controls. C4d deposition in PTC was found in 31 patients (6.81%) with LN. Patients with PTC C4d positive showed higher SLEDAI score and higher frequency of hypocomplementemia as compared to C4d negative. The prevalence of ANA, anti-dsDNA, anti-Sm and ACA were higher in C4d positive group, and most of these patients showed the diffuse proliferative glomerular lesion. In contrast with acute AMR, the staining pattern of C4d was granular deposition and the detection of C4d along PTC was accompanied by deposition of IgG and C1q or C3. Electron dense deposition on PTC was observed in most of LN patients. In conclusion, C4d deposition in PTC could be found in a small part of patients with LN. Our study suggested for the first time that C4d positive deposition were close relation with the higher disease activity of LN, and that immune complex formation might be involved in PTC C4d deposition in LN patients, Such PTC C4d deposition is distinct from that of AMR.