Samstag, 03.10.2020

14:00 - 15:30

Poster DGfN 2020

Chronisches Nierenversagen 4 (P145 - P153)

 
Das therapeutische Potential von Zinc-alpha2-Glycoprotein (AZGP1)
P145 

I. Sörensen-Zender, R. Schmitt; Hannover

Hintergrund: Die chronische Nierenerkrankung (CKD) zeichnet sich durch einen langfristigen Verlust der Nierenfunktion aus. Zinc-alpha-2-Glykoprotein (AZGP1, ZAG) ist ein 41 kDa großes, sezerniertes Glycoprotein, das in vielen verschiedenen Geweben exprimiert wird. Die Serumkonzentration von AZGP1 steigt bei Patienten mit Nierenfunktionsverlust deutlich an. In AZGP1 defizienten Mäusen konnten wir zeigen, dass AZGP1 protektive Eigenschaften bezüglich CKD-Progression und Herzfibrose aufweist. Im aktuellen Projekt sollte das Therapiepotenzial einer experimentellen AZGP1 Erhöhung für CKD Progression getestet werden.
Methode: C57BL/6 Mäuse wurden mit rekombinantem AZGP1 behandelt und es wurde ein neuer transgener Mausstamm generiert, welcher AZGP1 konditionell, tubulär überexprimiert. Unilaterale Ureterobstruktion (UUO) und Aristolochinsäure (AA) induzierte Nephropathie wurden als Schadensmodelle verwendet und die Nieren mittels Histomorphologie und Schadensmarkerexpression zu verschiedenen Zeitpunkten untersucht.
Ergebnisse: Die zusätzliche Gabe von rekombinantem AZGP1 oder die tubuläre Überexpression von AZGP1 verbesserte das renale Schadensmuster in vivo. Die protektive Wirkung zeigte allerdings eine starke Dosis-Abhängigkeit, welche sich in dem Modell der konditionellen Überexpression besonders zeigte. Im Nierengewebe von AZGP1 überexprimierenden Mäusen konnten im Vergleich zu Kontrollmäusen nur auf RNA-Ebene Unterschiede in AZGP1-Levels nachgewiesen werden, während im Serum deutlich erhöhte Level gefunden wurden. In Bezug auf Schadens- und Fibrosemarker zeigte sich in den transgenen Mäusen eine leichte Verbesserung, welche allerdings variabel war und nur in wenigen Markern Signifikanz erreichte. In mit rekombinanten AZGP1 behandelten C57BL/6 Mäusen zeigte sich die protektive Wirkung von AZGP1 deutlicher, wobei hohe Dosen bis zu 6x 150 µg i.v. injiziert wurden.
Zusammenfassung: Unsere Resultate bestätigen eine protektive Wirkung von AZGP1 im UUO Modell. Der Effekt ist jedoch moderater als vermutet. Während AZGP1 Knockout Mäuse einen deutlich aggravierten profibrotischen Phänotyp zeigten, hatte die therapeutische Steigerung von AZGP1 durch transgene Überexpression oder durch exogene Applikation eine zwar positive dosisabhängige Wirkung aber nur geringe Effektstärke.

 
Renal volume and association with risk factors for chronic kidney disease in a general population - an MRI based study
P146 

T. Dabers, P. Saß, J. Weyer, F. Fechner, R. Kuschnereit, S. Freiin von Rheinbaben, T. Petsch, R. Lorbeer, B. Mensel, S. Stracke; Greifswald, München, Jena

Objective: We assessed the renal volume (RV) and analyzed associated risk factors for chronic kidney disease (CKD) in the general population of Western Pomerania using magnetic resonance imaging (MRI).
Method: MRI-based determination of RV was performed semi-automatically for 1,815 participants of the population-based Study-of-Health-in-Pomerania (SHIP). RV according to age, sex, and glomerular filtration rate were analyzed by using fractional polynomial regression models, associations of renal volume and risk factors by multivariable linear regression.
Results: In every age group female kidneys were smaller than male kidneys, even when indexed to body surface area (BSA; p<0.001). Females showed a decreasing renal volume with increasing age, whereas males had the highest renal volume in the age group 40-59 years. RV of left kidneys are larger than right kidneys. Men had a lower proportion of never-smoker (31 vs. 47%) and a higher proportion of hypertension (50 vs. 37%) than females. Renal volume was positively associated with estimated glomerular filtration rate (p<0.001), current smoking (p<0.001), BSA (p<0.001) and diastolic blood pressure (p<0.05).
Conclusion: We provide reference values for MRI-based RV. As RV is strongly associated with estimated glomerular filtration rate, which itself is an important prognosis parameter for chronic kidney disease, MRI-based renal volumetry could be a valid tool in diagnosing and follow-up of renal diseases.

 
Protein-Signature of Early Cyclo sporin A-induced Renal Injury
P147 

R. Labes, L. Brinkmann, S. Mathia, B. Balcerek, V. A. Kulow, K. Spasova, P. B. Persson, C. Rosenberger, M. Fähling; Berlin

Hintergrund: Cyclosporin A (CsA) helps prevent allograft rejection and treat autoimmune diseases. But, such benefit sometimes is at the expense of renal toxicity. In previous studies, we have demonstrated that repeated CsA treatment results in episodic renal hypoxia. Here we investigated early mechanisms of the development of chronic kidney disease caused by CsA. In addition, we investigated the effects of daprodustat, a HIF-prolyl hydrolase inhibitor currently under investigation for treatment of renal anemia.
Methode: Mice received daily doses of either vehicle, daprodustat, CsA, or CsA/daprodustat for 8 weeks, after which kidney samples were collected. Samples were analyzed by Sciomics GmbH using antibody microarrays targeting 1360 different proteins at expression, as well as phosphorylation level. Proteins with a relative |logFc| bigger than 0.25 and an adjusted p-value smaller than 0.05 were considered significantly regulated. Pathway analysis was carried out using Gene set enrichment analysis (GSEA) according to the SetRank method.
Ergebnisse: Following treatment of CsA, 82 proteins were differentially expressed and 90 proteins changed their phosphorylation status compared to the sham group. GSEA revealed 68 (protein expression) and 71 (phosphorylation status) biological processes that may play a role in CsA toxicity. When combining daprodustat and CsA treatment, 49 proteins were differentially expressed and 31 proteins changed their phosphorylation status compared to CsA alone. Regarding the GSEA 56 (protein expression) and 71 (phosphorylation status) biological processes changed through the co-treatment with daprodustat. Among these processes, we found that CsA induced pathways linked to fibrosis, such as the TGF-beta pathway, are suppressed by Daprodustat.
Zusammenfassung: Proteomic analysis of 1360 proteins at expression as well as phosphorylation level revealed that 1) the CsA induced change in renal proteome is largely modulated by the HIF activator daprodustat; 2) daprodustat not only alters protein expression, but also strongly affects protein phosphorylation; and 3) daprodustat suppresses CsA induced pro-fibrotic factors. Daprodustat may help ameliorate CsA induced renal fibrosis.

 
NK cell-dependent tissue protection in crescentic glomerulonephritis
P148 

A.-C. Gnirck, C. Rickassel, T. F. Koyro, C. Körner, T. B. Huber, G. Gasteiger, U. Panzer, J.-E. Turner; Hamburg, Würzburg

Objective: Natural killer (NK) cells represent specialized effector lymphocytes of the innate immune system and are strongly involved in the regulation of immune responses. With respect to the kidney, understanding of NK cell function is still very limited. It has become evident that NK cells can be subdivided into conventional (circulating) NK (cNK) and tissue-resident NK (trNK) cells defined by unique surface marker and transcription factor profiles. However, the differential functions of these subsets, especially in the context of immune-mediated diseases, remain to be elucidated. Here, we investigated phenotypical properties and performed functional studies of the NK cell subsets in kidney tissue under homeostatic and inflammatory conditions.
Method: For NK cell characterization in mice, we applied a combination of flow cytometric analyses and bulk RNA sequencing. In order to assess subset-specific NK cell function, we used antibodies directed against the NK cell receptor NK1.1 for total NK cell depletion, anti-asGM1 antibodies for “selective” depletion of conventional NK cells, as well as Hobit-/- mice showing reduced numbers of trNK cells. The effects of different depletion strategies on disease outcome were analyzed in a mouse model for crescentic glomerulonephritis (cGN). Human NK cell populations were isolated from paired kidney and blood samples of patients undergoing tumor nephrectomy and phenotyped by flow cytometric analyses.
Results: Our results demonstrate that in addition to CD49b+T-bet+Eomes+CD127- cNK cells, around 25% of the total NK cell population in the kidney are CD49a+T-bet+Eomes-CD127+ trNK cells, resembling ILC1s. RNA sequencing data underline substantial transcriptional differences of the two distinct NK cell subpopulations and identify potential mediators of trNK cell function in the kidney. In a mouse model for cGN, depletion of total NK cells or cNK cells resulted in a significant increase of histopathological kidney damage, while trNK cell-impaired Hobit-/- mice did not show a substantial difference in disease outcome. Importantly, in the healthy human kidney conventional NK cells as well as tissue-resident CD69+CD49a+CD103+/- NK cells could also be identified.
Conclusion: Our preliminary data indicate a distinct trNK cell subset in the murine and human kidney and suggest a regulatory role of cNK cells in a mouse model of crescentic GN. Further investigations are aimed at elucidating the functional role of trNK cells in the kidney, as well as the molecular pathways that cNK cells employ to protect from immune-mediated glomerular damage.

 
Analysis of pneumococcus IgG antibody levels in HD patients applying a regular three-year revaccination regime with polyvalent vaccine: single-center cohort study.
P149 

K.-A. Brensing, P. Heidkamp, T. Gerhardt, U. Pöge, P. Raab; Bonn

Objective: Hemodialysis (HD) patients are at high mortality risk due to infection and/or cardio-vascular events. The need for sufficient immunization against serious respiratory infections as pneumococcus was reconfirmed by recent guidelines (Robert-Koch-Institute - August 2019) on new conjugate vaccine (Prevenar-13) for high-risk populations, but valid immune data on german HD cohorts is still missing. We evaluated immune status in a HD cohort treated with a regular 3-yr re-vaccination (Vac) regime using polysaccharid vaccine since 2004.

Method: We analysed global IgG antibody level against pneumococcus (mg/L, range 5-275 mg/L) in 158 chronic HD-patients prior to last winter season (11/19) from a routine blood sample at least 4 months after last polyvalent Vac (130x Pneumovax-23, 8x Prevenar-13, 19x no Vac). In general population the IgG level is in 95% higher than 10 mg/L and in 35% higher than 90 mg/L. We applied this higher IgG antibody level as limit for effective and a level <30 mg/L as limit for insufficient Vac response.

Results: Our cohort (male: 102; female: 56) had a mean (SD) age of 62 (16) yrs and vintage time was 5.5 (5) yrs (range: 0,3 – 27 yrs). Overall we saw a median (range) antibody level of 127 (9-275) after median of 1 Vac (0-5). This IgG level was significant lower without Vac (n=19; 12%: 73 (18-275) than after 1 Vac (n=61; 39%: 138 (19-275), 2 Vac (n=29; 18%: 135 (9-275) or after 3-5 Vac (n=49; 31%: 128 (13-275), (p<0.01). Median IgG increase without Vac compared to with at least one Vac was 85% (73 to 135 mg/L).
High IgG levels (>90 mg/L) were measured in 9/19 (47%) without Vac, 38/61 (62%) after 1 Vac; 18/29 (62%) after 2 Vac and 33/49 (67%) after 3-5 Vac. Low IgG levels (<30 mg/L) had 7% (11/158): 2/19 (11%) without Vac.; 3/61(5%) after 1 Vac., 3/29 (10%) after 2 Vac. and 3/49 (6%) after 3-5 Vac. Thus, from all pts. with at least one Vac. 64% (89/139) had high antibody level > 90 mg/L and 6,5% (9/139) were non-responders (<30 mg/L). Our routine Vac regime achieved an overall Vac rate of 88% (138/158) and of 93% in patients with a vintage > 1yr (138/149). Finally, Vac was very well tolerated and accepted for Re-Vac request.

Conclusion: Regular Vac with polysaccharide vaccine achieved good Vac rates of 90% and induced a high immune response in 2/3 of HD patients for 10-15 yrs. Non-responder rate is rather low (7%) after at least one Vac. How IgG levels are related to effective prevention of severe pneumococcus infections like pneumonia and if the new conjugate vaccine (Prevenar-13) can improve immune status should be evaluated in future trials.

 
Systematische Reevaluation genetischer Befunde von Patienten mit Alport-Syndrom/Syndrom der dünnen Basalmembran zeigt hohe Rate an nicht eindeutigen Ergebnissen
P150 

K. Riedhammer, P. Richthammer, D. Westphal, J. Comic, R. Günthner, M. Braunisch, S. Rath, A. K. Büscher, H.-G. Klein, S. Weber, J. Höfele; München, Planegg, Essen, Marburg

Hintergrund: Das Alport-Syndrom (AS) ist eine hereditäre Nierenerkrankung, die mit Mikrohämaturie und Nierenfunktionsverlust einhergeht. AS wird X-chromosomal (COL4A5, 80-85%) und autosomal-rezessiv (COL4A3/COL4A4, ca. 15%) vererbt. Die Bezeichnung "autosomal-dominantes AS" (ADAS) ist umstritten, da heterozygote Varianten in COL4A3/COL4A4 typischerweise zu einem milderen Phänotyp mit reduzierter Penetranz [Syndrom der dünnen Basalmembran (TBMN)] führen. Varianten werden seit 2015 durch Kriterien des American College of Medical Genetics (ACMG) bewertet.
Methode: Es wurden genetische Befunde von 97 Indexfällen mit der Verdachtsdiagnose AS (77/97, 79%), TBMN (14/97, 14%) oder unklarem Phänotyp [AS/TBMN (6/97, 6%)], die zwischen 2009 und 2014 eine Untersuchung der COL4A3-5-Gene bekamen, reevaluiert. Alle berichteten Varianten wurden mittels ACMG-Kriterien reinterpretiert. Varianten mussten sowohl auf Varianten-, als auch auf Genotypebene (Kombination/Zygotie der Varianten) ursächlich sein, damit der Fall als „gelöst“ bezeichnet wurde. Krankheitsverursachende Varianten, die lediglich heterozygot in COL4A3/COL4A4 bei einem AS-Fall vorlagen, waren auf Genotypebene nicht erklärend. 7/97 (7%) Fälle konnten wegen fehlender Daten bei der Auswertung "Fall gelöst ja/nein" nicht berücksichtigt werden.
Ergebnisse: Es wurden 85 Varianten (verteilt auf 97 Fälle) reinterpretiert: 21/85 (25%) in COL4A3, 18/85 (21%) in COL4A4 und 46/85 (54%) in COL4A5. Bei den 90 Fällen, die für die weitere Auswertung berücksichtigt werden konnten (siehe Methoden), waren 83/90 (92%) ursprünglich als gelöst befundet, nach Reevaluation nur noch 64/90 (71%) (p<0.001, Fisher-Exact-Test). 15/90 (17%) Fälle waren nach Reevaluation auf Variantenebene nicht gelöst, 8/90 (9%) nicht auf Genotypebene, 3/90 (3%) weder auf Varianten-, noch auf Genotypebene (Abbildung 1).

P150

Zusammenfassung: Für behandelnde Ärzte ist eine nachvollziehbare Varianten-/Genotypinterpretation entscheidend, um hereditäre Erkrankungen zu diagnostizieren, Therapien festzulegen, Prognosen abzuschätzen und Wiederholungsrisiken anzugeben. Mit dieser Studie konnte bei systematischer Reevaluation von genetischen Befunden auf Varianten-/Genotypebene eine signifikante Reduktion der Diagnoserate von AS/TBMN gezeigt werden. Befunde, die vor 2015 erhoben wurden (Einschätzung der Pathogenität mittels vorhandener Literatur und in-silico-Vorhersageprogrammen) und/oder mit der Diagnose ADAS, sollten kritisch hinterfragt werden.

 
Prevalence of hereditary diseases with a fibrotic, tubulointerstitial phenotype in the German Chronic Kidney Disease cohort
P151 

B. Popp, A. Ekici, K. Knaup, S. Uebe, K. Schneider, K.-U. Eckardt, GCKD consortium, M. Schiffer, A. Reis, M. Wiesener; Leipzig, Erlangen, Berlin

Objective: Screening studies applying exome sequencing (ES) in cohorts with chronic kidney disease (CKD) could identify pathogenic variants in about 10%. This implies hereditary CKD being underdiagnosed. Tubulointerstitial kidney diseases are particularly difficult to diagnose as they show no typical clinical signs and no histologic finding other than tubulointerstitial fibrosis. Genetic disorders typically associated with a fibrotic, tubulointerstitial phenotype are autosomal dominant tubulointerstitial kidney diseases (ADTKD), mitochondrially inherited tubulointerstitial kidney diseases (MITKD), nephronophthisis (NPHP) and often Collagen 4 (COL4) diseases.
Method: Based on the GCKD (German CKD) cohort we filtered for different criteria including age, sex, and suspected renal disease to identify 304 individuals as candidates for hereditary tubulointerstitial kidney diseases. We used a targeted panel including 29 genes, the variable number tandem repeat (VNTR) in MUC1 and the mitochondrial genome to sequence 271 DNA samples passing stringent quality tests. To screen for the cytosine insertion causing ADTKD-MUC1 we used an established probe extension (SnaPshot) assay.
Results: We identified 32 pathogenic point mutations in 30 patients (11.1%). Of these 26 (81.3%) were in the COL4 genes, the largest group being 15 COL4A5 variants with 9 unrelated individuals carrying c.1871G>A, p.(Gly624Asp). We found three typical cysteine mutations in the UMOD gene, a novel missense, and a novel splice-site mutation in HNF1B and the homoplastic transition m.616T>C in MTTF. One individual carried variants in both COL4A5/COL4A3 and a second carried mutations in COL4A5/HNF1B. Coverage based copy-number (CN) analysis identified a heterozygous intragenic COL4A5 deletion, and a duplication and a deletion of HNF1B, respectively. Overall, we confirmed mutations in 12.2% and found variants of unknown significance in 9.6%.
Conclusion: Our study confirms and extends recent findings and shows that clearly more than 10% of individuals with certain clinical features carry disease variants in genes associated with tubulointerstitial kidney disease. Type IV collagen genes constitute the largest fraction, implying that this group is largely overlooked applying current diagnostic criteria for Alport syndrome. We identified a mitochondrial variant and 3 CN-variants which could be missed by typical ES pipelines. Snapshot could not confirm the predictions of bioinformatic algorithms used to detect variants in the MUC1-VNTR. We are now analyzing all samples using this gold standard to establish the prevalence of ADTKD-MUC1.

 
Comparison of the renal effects of heme-dependent and independent sGC targeting drugs in 5/6 nephrectomized rats on high salt diet
P152 

Y. Xiong, S. Zeng, A. Hasan, D. Delic, M. M. S. Gaballa, M. Li, M. Pavkovic, P. Sandner, J.-P. Stasch, B. K. Krämer, L. Yin, B. Hocher; Potsdam, Mannheim, Toukh/ET, Wuppertal, Guangzhou/CN

Objective: Soluble guanylate cyclase (sGC) targeting drugs were reported to have beneficial renal effects in chronic kidney disease (CKD). The sGC stimulators bind to reduced, heme-containing sGC, while sGC activators bind to oxidized, heme-free sGC and their actions are heme-independent. Regarding renal outcomes, the potential differences between these two classes of drugs are unknown so far. The present study aimed to provide a head-to-head comparison of the renal effects of BAY 41-8543 (sGC stimulator) and BAY 60-2770 (sGC activator) in 5/6 nephrectomized rats on high salt diet as a model of CKD
Method: Rats were allocated to the following groups: Sham + normal diet + placebo (PBO); 5/6Nx + 2 % high salt diet (HSD) + PBO; 5/6Nx + HSD + Telmisartan (5mg/kg/day) an angiotensin II receptor blocker (ARB) as a the gold-standard treatment of CKD; 5/6Nx + HSD + BAY 60-2770 (1 mg/kg/day); 5/6Nx + HSD + BAY 41-8543 (1mg/kg/day). The treatment period was 8 weeks
Results: Blood pressure was significantly decreased by BAY 60-2770 and BAY 41-8543 versus placebo (-32.52/-27.20 mmHg, p<0.001; -23.83/-29.90 mmHg, p<0.001, respectively), which was also comparable to the effects of telmisartan (-24.24/-31.90 mmHg, p<0.001). Plasma creatinine was not altered by any of the 3 drugs, however, renal fibrosis was significantly decreased by the sGC activator Bay 60-2770 (44.76%, p<0.05) and telmisartan (43.96%, p<0.05) versus placebo. On the other hand, the sGC stimulator BAY 41-8543 did not ameliorate renal fibrosis.
RNA-sequencing in renal tissues was performed as an open approach strategy to investigate the mechanisms underlying these effects. RNA-sequencing revealed that 144 genes were differentially regulated among the groups. Interestingly, 23 genes including collagen type VI alpha 5 (Col6a5), phospholipase C Eta 1  (Plch1) and claudin 19 (Cldn19) were exclusively differentially regulated by the sGC activator BAY 60-2770 and these genes might be involved in the mechanisms underlying its anti-fibrotic renal effects
Conclusion: The heme-independent sGC activator BAY60-2770 ameliorated renal fibrosis comparable to the gold-standard treatment of CKD with an ARB (telmisartan). These effects were blood pressure independent since blood pressure was similar in all treatment groups. Inactivation of the sGC by oxidative stress in our CKD model may explain the failure of the sGC stimulator in reducing kidney fibrosis. The mechanisms underlying the renal anti-fibrotic effects of the sGC activator BAY60-2770 might involve the differential regulation of Col6a5Plch1 and Cldn19

 
C3 Glomerulopathy: Using Serum of a C3G patient with a new Factor H gene mutation for in vitro testing of complement inhibitors and modulators
P153 

C. F. Krebs, S. Afonso, E. Decker, C. Skerka, T. Wiech, P. F. Zipfel; Hamburg, Jena, Freiburg

Objective: C3 Glomerulopathy is a rare, chronic kidney disease that ultimately damages the glomerular structure. C3 glomerulopathy is caused by defective complement and the causes are heterogenous. Genetic defects, as well as circulating auto-antibodies lead to overactivation and deregulation of complement in plasma and on glomerular surfaces.
Method: Serum of a C3G patient was evaluated for complement parameters such as C3 and activation markers like Ba and TCC/sC5b-9. In addition genetic analyses and kidney biopsy were performed. In vitro complement tests in form of hemolytic assays of patient serum and cell damaging assays were performed and complement inhibitors and modulators were assayed in vitro.
Results: We identify a C3G patient with low C3 plasma levels and high levels of activation products Ba and TCC/sC5b-9. Genetic analyses revealed a novel, heterogenous mutation in the Factor H gene, which affects serum levels of both Factor H and FHL1, the alternatively spliced gene product. Renal biopsy showed C3b deposition, double contours of the glomerular basal membrane and the presence of alternative pathway convertases. Serum of the patient was used to evaluate complement activity in vitro hemolytic assays and to quantitate complement damage. In this system the action of complement inhibitors such as Eculizumab, Danacopan, Compstatin, sCR1 as well as recombinant moss produced Factor H were evaluated and compared. Showing that complement inhibition at the level of the C3- and the C5 convertase blocked lytic complement action.
Conclusion: We identify a C3G patient who has a new mutation in the Factor H gene. Renal biopsies showed strong s formation of the C3 convertase of the alternative pathway. Furthermore we established and used in vitro assays to evaluate and compare the efficiency of various complement inhibitors and modulators aiming to select which type of complement inhibition may be best suited for this patient.

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Fragen und Antworten der Posterbewerter und Autoren

Kommentar von Vedat Schwenger |

P 147: Innovatives Tiermodell, aufbauend auf Voruntersuchungen (Tiermodell= C57BL/6?). HIF-Aktivator Daprodustat scheint zumindest auf Genebene protektiv vor CsA induzierter renaler Schädigung zu sein. Interessant wäre hier das histologische read out, bzw. histologische Korrelat (Phänotyp?).

Kommentar von Vedat Schwenger |

P 146: interessante klinische Untersuchung im Hinblick auf "gender" spezifische Unterschiede, ggf. auch im Hinblick auf altersabhängige Donornephrektomie. Unklar, bzw. überraschend ist, warum "current smoking" mit renal volume positive korreliert ist?

Kommentar von Vedat Schwenger |

P 145: Auch wenn die Ergebnisse nicht so ausgefallen sind wie erhofft, methodisch sehr gute Arbeit auf hohem Niveau. Sowohl Tiermodell, als auch Schädigungsmodell sind etabliert. Sehr interessantes transgenes Tiermodell. Wissenschaftlich "seriöse" und adäquate Diskussion der Befunde

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