Samstag, 03.10.2020

14:00 - 15:30

Poster DGfN 2020

Transplantation 4 (P229 - P236; LA48)

 
Immunosuppression by cyclosporine A affects proximal tubular homeostasis: roles of endoplasmic reticulum stress and autophagy
P229 

S. Popovic, J. Hu, H. Demirci, D. E. Yilmaz, C. Dittmayer, Y. Xu, M. Thomson, K. Mutig, S. Bachmann; Berlin

Objective: Calcineurin inhibitors (CNI) such as cyclosporine A (CsA) or tacrolimus are first-line immunosuppressive drugs used after solid organ transplantation. However, renal side effects such as vascular and tubulo-interstital malformations regularly occur in their long-term usage, especially in patients with renal allografts. Measures to reduce CNI nephrotoxicity by RAS inhibition or calcium antagonism had limited success. Dysfunctions in renal epithelial proteostasis suggested CNI-induced ER stress and malfunction of the unfolded protein response (UPR).
Method: We have established a rat model for chronic CNI nephrotoxicity to test whether epithelial pathology and loss of functioning nephrons is related to ER stress and UPR dysfunction. Adult male Wistar rats received cyclosporin A (CsA, 25 to 40 mg/kg. b.w.) or vehicle via subcutaneously implanted minipumps. After three weeks, rats were sacrificed and kidneys removed for NGS and proteomics, or perfusion-fixed and analyzed for histopathology.
Results: Rats with CsA displayed a stimulated RAS and increased distal NaCl transporter activity along with decreased urine volume, GFR, FENa, and COX-2 expression. Pathological changes in vasculature and glomeruli were inconspicuous, whereas early proximal tubular segments (S1, S2) revealed large lysosomal vacuoles with granular content, their abundance correlating with epithelial dedifferentiation, basement membrane thickening, and subepithelial collagen I accumulation. Protein endocytosis was diminished. Changes in UPR and autophagy parameters included pEIF2a, pPERK, CHOP, BiP, and LC3B, ATG5, p62 products. Parallel studies in cultured cells indicated sensitivity to chemical chaperones ameliorating proteostasis.
Conclusion: These results suggest a so far unrecognized role of proximal tubular homeostasis in long term CsA-induced nephrotoxicity. Addressing UPR, autophagy, and restitution of proteostasis in proximal tubule may, therefore, have renoprotective potential.

 
Deep Learning-Bildanalyse zur histopathologischen Erkennung einer antikörpervermittelten Abstoßung an Glomerulusquerschnitten
P230 

P. A. Cicalese, A. Weidemann, B. Schröppel, C. Kurschat, E. Leenen, L. T. Weber, W. Arns, H. V. Nguyen, J. U. Becker; Houston/USA, Köln, Ulm

Hintergrund: Die histopathologische Diagnose einer antikörpervermittelten Abstoßung (ABMR) ist unter anderem durch eine ungenügende Interobserver-Reliabilität erschwert. Bildanalyse mit Deep Learning-Methoden hat bereits in vielen Feldern überzeugenden Ergebnisse mit perfekter Reproduzierbarkeit geliefert. Daher wollten wir diese Methode zur Diagnose einer ABMR anhand von Glomerulusquerschnitten erproben.
Methode: Aus unserem Archiv wurden 48 Biopsien mit und 38 Biopsien ohne ABMR nach Banff 2017 für die Studie herangezogen. Biopsien mit anderen Glomerulupathien wurden ausgeschlossen. Alle offenen (ohne Globalsklerose) Glomeruli von zwei PAS-Schnittstufen wurden mit dem 40er Objektiv fotografiert und danach randomisiert und geblindet von einem erfahrenen Nephropathologen als entweder "ABMR" (n=1001), "keine ABMR" (n=503) oder "inkonklusiv" (n=140) klassifiziert. Nach Vergrößerung dieses 1644 Bilder umfassenden Datensatzes durch geringe Textur- und Farbänderungen wurde ein neuronales Netzwerk (das auf dem ImageNet-Datensatz prätrainierte DenseNet 121) einem überwachten Training bezüglich der Klassifikation unterzogen. Die Leistung des Netzwerkes wurde durch interne Kreuzvalidierung überprüft, wobei Glomerulusquerschnitte ein und derselben Biopsie entweder in der Trainings- oder der Testgruppe, aber niemals in beiden blieben. Die für die Klassifikation entscheidenden Areale wurden mit Grad-Cam als überlagerte Heatmap visualisiert.
Ergebnisse: Die Klassifikation des Nephropathologen konnte mit einer Genauigkeit von maximal 88,5% reproduziert werden. Durch die Visualisierung der für die Klassifikation entscheidenden Areale mittels Grad-CAM konnten viele der durch das Netzwerk fehlklassifizierten Glomerulusquerschnitte leicht als solche erkannt werden.
Zusammenfassung: Die Deep Learning-Bildanalyse ist ein sehr vielversprechender Ansatz zur Verbesserung und Standardisierung der histopathologischen Nierentransplantatdiagnostik; dies insbesondere in Verbindung mit Visualsierungstechniken wie Grad-CAM, durch die Klassifikationsentscheidungen für den verantwortlichen Pathologen nachvollziehbarer werden. Wir hoffen, durch Vergrößerungen und Entitätserweiterungen der Trainingssets bald eine leistungsfähiges Hilfssystem für die Einordnung glomerulärer Befunde in der Transplantatdiagnostik entwickeln zu können.

 
Erfolgreiche simultane Doppel-Lungen- und Nierentransplantation bei einer Patientin mit atypischem hämolytisch-urämischen Syndrom mit Lungenbeteiligung
P231 

E. Seelow, D. Kemper, A. Kahl, F. Halleck, B. Globke, R. Öllinger, D. Grund, H. Müller-Redetzky, C. Tabeling, M. Witzenrath, C. Knosalla, V. Falk, K.-U. Eckardt, F. Hennig; Berlin

Hintergrund: Das atypische hämolytisch-urämische Syndrom (aHUS) ist eine seltene, potentiell lebensbedrohliche Regulationsstörung des alternativen Komplementwegs, die zu einer thrombotischen Mikroangiopathie mit Nierenversagen führt. In 20% der Fälle sind auch andere Organsysteme betroffen. Seit der Zulassung von Eculizumab, eines monoklonalen Antikörpers gegen den Komplementfaktor C5, hat sich die Prognose des aHUS deutlich gebessert, ebenso das Transplantatüberleben nach Nierentransplantation. Wir berichten hier von einer 32-jährigen Patientin mit einer heterozygoten pathogenen Variante im CFI-Gen und einer weiteren heterozygoten Variante unklarer Signifikanz im MCP-Gen.
Methode: Nach Geburt ihres zweiten Kindes manifestierte sich ein aHUS mit chronisch dialysepflichtigem Nierenversagen. Seither steht sie unter einer Erhaltungstherapie mit Eculizumab. 1,5 Jahre nach Erkrankungsbeginn kam es Infekt-assoziiert zu einem fulminanten Rezidiv des aHUS mit mikroangiopathischer Hämolyse, Thrombopenie, ZNS-Beteiligung und pulmonaler Beteiligung. Wegen einer konservativ nicht beherrschbaren Oxygenierungsstörung mit Rechtsherzversagen und einer pulmonal-arteriellen Hypertonie (PAH) mit halbsystemischen Pulmonalisdrücken wurde eine V-VA ECMO implantiert (veno-venoartierelle extracorporale Membranoxygenierung). Trotz effektiver Inhibition des Komplementsystems unter intensivierter Eculizumabtherapie sowie optimaler medikamentöser Therapie der PAH gelang keine Entwöhnung der wachen und kreislaufstabilen Patientin von der ECMO. Die Patientin wurde daher für eine simultane Doppel-Lungen- und Nierentransplantation gelistet, die drei Tage später unter Eculizumab-Therapie durchgeführt wurde.
Ergebnisse: Die ECMO konnte noch im OP explantiert werden, das Nierentransplantat zeigte Primärfunktion. 6 Monate nach Transplantation ist die Blutgasanalyse unter Raumluft ausgeglichen, die FEV1 der Patientin liegt mit 1,94 L bei 54% vom Soll, das Kreatinin beträgt 1,2 mg/dl. Es besteht bis zuletzt keine aHUS-Aktivität mehr.
Zusammenfassung: Unseres Wissens nach ist dies der erste Fall einer kombinierten Lungen- und Nierentransplantation bei einer Patientin mit einem aHUS mit Lungenbeteiligung.

 
Inhibition of T follicular helper cell formation and IL-21 production prevents antibody-mediated rejection in a kidney transplant model in rats
P232 

L. Steines, H. Poth, A. Schuster, K. Amann, B. Banas, T. Bergler; Regensburg, Erlangen

Objective: Antibody-mediated rejection (AMR) is the leading cause of late kidney allograft failure. We aimed to examine the formation of T follicular helper cells (Tfh) and expression of interleukin-21 (IL-21) in secondary lymphoid organs in a kidney transplant model of AMR.
Method: We used a MHC-mismatched rat kidney transplant (Ktx) model and applied low dose cyclosporine A (CsA 5mg/kg/d, “loCNI”) to allow donor-specific antibody (DSA) formation and rejection and high dose CsA (10 mg/kg/d,”hiCNI”) for non-rejection. We measured DSA/IgG subclasses by flow crossmatch, leukocyte subsets by flow cytometry, germinal centers (GC), T follicular helper cells (Tfh) and plasma cells, as well as IL-21 expression by immunofluorescence microscopy. Expression of T cell and B cell markers were analyzed by qRT-PCR. Allograft rejection was evaluated by an experienced nephropathologist.
Results: HiCNI prevented formation of DSA and antibody-mediated rejection. HiCNI strongly inhibited the formation of GC and activated Tfh, resulting in lower numbers of plasma cells and memory B cells. B cell activation signals expressed by activated Tfh, such as CD40 ligand and ICOS, were reduced. Similarly expression of the B cell activating cytokine IL-21 was strongly inhibited in hiCNI, but not loCNI, while other T cell cytokines were less profoundly affected.
Conclusion: Tfh formation and expression of IL-21 was strongly inhibited by hiCNI treatment, which completely prevented B cell activation in GC and development of DSA and AMR. Targeting Tfh is an effective strategy for preventing alloantibody formation and AMR.

 
Accuracy of genetic HLA-typing in living-donor kidney transplantation
P233 

S. Pehnke, B. Popp, A. Weimann, A. Bachmann, T. H. Lindner, I. Doxiadis, R. Landgraf, C. Lehmann, J. Halbritter, J. Münch; Leipzig

Objective: Antibody mediated rejection (ABMR) based on donor specific human leukocyte antigen (HLA) antibodies is the most common cause of long-term allograft loss in kidney transplant recipients. Therefore, a low count of HLA mismatches is favorable in the course of transplantation. Hitherto, several techniques for HLA-typing have been adapted in parallel, e.g. serological or low-resolution PCR. In this single-center study, we aimed to perform “HLA re-typing” by next generation sequencing (NGS) in a living kidney transplant cohort, in order to identify HLA mismatches in donor/recipient pairs, that were missed by previous HLA typing methods.
Method: We analyzed DNA of 103 donor/recipient pairs, having undergone living-donor kidney transplantation at a German transplant center between 1998 and 2018, by performing long range PCR of all known HLA loci. Using these data, we compiled a genomic HLA mismatch formula and comparatively evaluated genomic HLA-typing with HLA mismatches recorded in the Eurotransplant database (HLA-A, -B, C, -DQ, -DR).
Results: By genomic HLA re-typing, we were able to identify additional, previously missed HLA mismatches in 64.1% (n=66) of cases. The number of additionally detected HLA mismatches ranged from 1 to 5. In only 32.0% (n=33) of donor/recipient pairs, findings from genomic HLA-typing completely overlapped with previous results from (serological) HLA-typing. We identified 9 donor/recipient pairs with a hitherto unknown number of maximum mismatches in all 10 sequenced HLA loci. Unexpectedly, genomic HLA-typing showed less mismatches in 3.9% (n=4) donor/recipient pairs.

P233

Conclusion: Our results indicate that genomic HLA-typing is significantly more precise in detecting HLA mismatches. Especially in living-donor kidney transplantation, higher accuracy in pre-transplant HLA-typing might enhance adequate donor selection. Furthermore, accurate HLA typing can influence graft management in the course of ABMR, as it may improve discrimination between donor specific and unspecific HLA directed antibodies.

 
Association of BAFF-var polymorphism and rs9514828C>T polymorphism in the promotor of BAFF-encoding gene with infections and allograft failure after renal transplantation
P234 

J. Friebus-Kardash, F. Heinemann, E. Nela, B. Wilde, K. Hallmann, A. Kribben, U. Eisenberger, C. Hardt; Essen

Objective: B-cell activating factor (BAFF) activates alloimmune response by stimulation of alloreative B-cells and is involved in preimmunization and rejection in renal transplant patients. Functional polymorphism in the BAFF-encoding gene, BAFF-var, is associated with elevated production of soluble BAFF and increased susceptibility to autoimmune disorders. The purpose of present study is to investigate the prevalence of both polymorphism, BAFF-var and rs9514828C>T, in renal allograft recipients and to evaluate the association with BAFF expression pretransplant, renal allograft outcome and incidence of infections.
Method: A retrospective single-center study was conducted on 448 renal transplant patients and 200 healthy controls. We performed genotyping for BAFF-var allele using polymerase chain reaction. The rs9514828C>T polymorphism was determined by pyrosequencing. Serum levels of soluble BAFF were quantified by ELISA. Clinical follow-up was analyzed up to 5 years posttransplant.
Results: The BAFF-var variant was prevalent in 22 (5%) of all renal allograft recipients and in 12 (6%) control subjects. Renal transplant patients as well as controls carrying the BAFF-var allele had significantly increased pretransplant serum levels of BAFF compared with nonexpressers. Analysis of rs9514828C>T polymorphism among renal allograft recipients revealed 82 (18%) homozygous and 211 (47%) heterozygous patients. Analyzing pretransplant serum levels of BAFF according to the rs9514828C>T polymorphism, no significant differences were observed in patients and controls, respectively. However, concomitant appearance of BAFF-var with rs9514828 T variant allele was associated with significantly increased BAFF expression in recipients and controls. The 22 recipients carrying BAFF-var displayed significantly higher frequency of infectious complications posttransplant such as CMV viremia, CMV disease and pyelonephritis than nonexpressers. Indeed, homozygous patients for the rs9514828C>T polymorphism experienced more often allograft failure and a decrease in eGFR of more than 50% compared with heterozygous patients or patients without polymorphism during the follow-up. We did not see any influence of BAFF-var and rs9514828C>T polymorphisms on rejection events and development of de novo anti-HLA antibodies.
Conclusion: Recipients carrying BAFF-var combined with rs9514828C>T polymorphism had the highest expression of soluble BAFF. While BAFF-var was related to infections posttransplant, the rs9514828 T variant seems to contribute to allograft loss.

 
Individualized immunosuppression by monitoring of NFAT-regulated gene expression in calcineurin inhibitor treated renal transplant recipients
P235 

C. Sommerer, J. Brocke, S. Meuer, M. Zeier, T. Giese; Heidelberg

Objective: Calcineurin inhibitors are critical-dose drugs with a narrow therapeutic range and the optimal monitoring strategies are discussed in terms of safety and efficacy. A new pharmacodynamic monitoring tool – assessing the expression of nuclear factor of activated T cells (NFAT)-regulated genes – has been established to measure directly the functional effect of calcineurin inhibitors (CNI) in an individual patient.
Method: The expression of the NFAT-regulated genes (RGE; interleukin 2, granulocyte-macrophage colony stimulating-factor, interferon y) was determined by qRT-PCR at Ciclosporin A (CsA) C0 and C2 in whole blood samples. Intraindividual and interindividual variability of RGE was assessed. For interlaboratory comparison, samples of 20 patients were analyzed in two different laboratories.
Results: Altogether, 362 renal allograft recipients on CsA therapy were enrolled (63% male, age 51.3 (17-78) years, time after transplantation 47.3 months, 30.9% living donation). NFAT-RGE showed a high interindividual variability, but a low intraindividual variability. Interlaboratory comparison in 20 patient samples correlated well (r=0.970, p<0.001).  Mean NFAT-RGE increased from 5% at month 6 to 18% 10 years after transplantation. Patients with an NFAT-RGE ≥ 20% were on increased risk for biopsy-proven acute rejection (OR 3.98, CI 1.24-12.79), p=0.02), but at significantly lower risk for infectious events (OR 0.09, CI 0.01-0.73), p=0.002). Only 8 patients with NFAT-RGE ≥ 20% developed non-melanoma skin cancer (OR 0.42, CI 0.15-1.16, p=0.08).
Conclusion: NFAT-RGE provides the opportunity to individualize CsA treatment in renal allograft recipients. NFAT monitoring supports pharmacokinetics and is suggested as an additional monitoring tool to optimize CNI treatment.

 
Living renal donor characteristics over the past five decades – a single center analysis
P236 

C. Sommerer, Z. Bougioukou, V. L. Georgiou, C. Morath, M. Zeier; Heidelberg, Patra/GR

Objective: In view of a changing landscape in renal transplantation selection criteria for potential living donors may alter. The objective of this study was to evaluate the shift of living kidney donor characteristics during the last decades.
Method: Baseline characteristics of 765 living kidney donors from 1967 to 2016 were analyzed.
Results: Mean donor age increased significantly from 34.9±11.5 to 53.2±10.2 years. Living donors aged > 65 years enlarged from 0% to 11.4%. The prevalence of co-morbidities at the time of donation increased. Percentage of obese donors and hypertensive donors rose up to 18.6% and 37.0%, respectively. 37% of the donors were active smokers. Renal function prior to donation was stable. None of the patients presented significant proteinuria or pre-existing diabetes.
Conclusion: A detailed analysis of donor characteristics of living kidney donors over the past 5 decades revealed increasing age, body mass index, number of co-morbidities in the donor cohort. Clinical follow-up data should be analyzed carefully to evaluate donor and recipient outcome.

 
Study protocol of an observational, prospective, multi-center cohort Study of kidney TRAnsplant BIOpsies (TRABIO) for suspected graft rejections
LA48 

F. A. von Samson-Himmelstjerna, G. Esser, K. Schulte, B. Kolbrink, T. Feldkamp, U. Kunzendorf, L. Renders, U. Heemann; Kiel, München

Objective: Acute and chronic kidney graft rejections are strong independent risk factors of graft failure. Use of immunosuppressants for induction, maintenance and rejection therapy has improved long-term outcomes drastically, and implementation of the Banff-classification in 1993 has widely standardized diagnostic evaluation. Yet, graft rejections continue to pose a major challenge to transplantation centers around the world and long-term graft survival has not been improved in about 20 years. Unfortunately, data on pathohistology, outcomes and treatment strategies of acute rejection episodes in Germany to guide clinical decision-making are scarce. We have launched a prospective registry of TRAnsplant BIOpsies (TRABIO) in six transplantation centers for evaluation of the current situation in Germany.

Method: In this observational, prospective, multi-center cohort study, we intend to enroll 800 kidney transplantation patients undergoing indication kidney biopsy for suspected episodes of acute graft rejection. Recruitment was initiated in April 2017 and 495 patients have been enrolled as of August 2020. The primary outcomes of interest are mortality and new-onset end-stage renal disease, and the secondary outcomes are worsening of glomerular filtration rate and re-biopsies for suspected recurrence of graft rejection. After informed and written consent has been acquired, demographic and medical baseline data are registered and entered into an electronic data portal. During a first follow-up within 14 days, detailed biopsy findings, laboratory results, treatment strategy and clinical course are recorded. Patients are followed-up after 12 and 24 months for repeated assessment of baseline medical data, clinical course and treatment outcomes. Description of the registry population will be conducted using counts with percentages for categorical or binary variables, and means with standard deviations for continuous variables. Logistic and linear regression analyses will be used to analyze association of outcomes with treatment strategies and Banff-categories. The registry is funded by Chiesi GmbH and monitored by Gesellschaft für Therapieforschung mbH. Ethical approval was given by each participating center’s ethics committee (AZ B 278/16).

Results: We present the study protocol of the TRABIO registry. Final results will be reported in a peer-reviewed journal, using the Strengthening the Reporting of Observational Studies in Epidemiology criteria.

Conclusion: Findings of the TRABIO registry will provide a data basis for future updates on national guidelines concerning kidney graft rejections.

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