Freitag, 02.10.2020

14:00 - 15:30

Poster DGfN 2020

Experimentelle Nephrologie 1 (P058 - P066; LA13 - LA15)

 
Ein dreidimensionales „Kidney-on-the-chip“-Modell mit primären humanen renalen Progenitorzellen, Endothel und Immunzellen
P058 

J. Marschner, G. Wilken, P. Romagnani, H.-J. Anders; München, Florence/I

Hintergrund: Zellkulturmodelle präklinischer Nierenforschung bestehen in der Regel aus Einzelzellsystemen immortalisierter Zelllinien in 2-dimensionalem Design. In Pharmakologie und Toxikologie bilden diese Modelle oft nur unzureichend in-vivo beobachtete Effekte ab. Komplizierte und aufwändige Protokolle limitierten jedoch den Einsatz komplexerer Techniken in der Routinezellkultur. Unser Ziel ist daher die Etablierung und Validierung eines 3D-Kokulturmodells eines renalen Tubulussegmentes aus Tubuluszellen, peritubulärer Kapillare und interstitiellem Kompartiment.
Methode: Grundlage unseres Modells bildete das 3-Kanal OrganoPlate-System (Mimetas, Leiden, Niederlande).

P058

Nach Implementierung von Kollagen-I in den mittleren Kanal (C2) wurden primäre humane renale Vorläuferzellen in den oberen Kanal (C1) eingebracht. Nach Adhäsion der Zeleln an der C2-Matrix wurde ein kontinuierlicher bidirektionaler Fluss im gesamten Chip induziert, was innerhalb von 48h zur Ausbildung einer intakten epithelialen Barriere der tubulären Struktur mit durchgängigem Lumen führte. Anschließend wurden endotheliale Zellen in den unteren Kanal (C3) gesät, welche auf die gleiche oben beschriebene Weise eine lumenhaltige 3-dimensionale Konformation aufweisen. Die Implementierung humaner primärer Leukozyten (Neutrophile/ Lymphozyten/ Monozyten) in die kapilläre Struktur (C3) komplettierte das Modell.
Ergebnisse: Die Etablierung der Kokulturen aus Epithel und Endothel dauerte in etwa 3 Tage. Über die Messung der Diffusionsgeschwindigkeit eines Dextran-Tracers (150 kD) zeigte sich eine stabile Barriereintegrität über 5 Tage. Des weiteren konnte im Vergleich zu 2D-Zellkulturen über Immunfluoreszenzfärbung eine höhere Expression funktionell wichtiger Proteine (Na+-K+-ATPase, ZO-1, F-Aktin) gezeigt werden. Die Schädigung des Tubulus mit extrazellulären Histonen ermöglichte die Abbildung eines pathophysiologisch relevanten Prozesses, wie er bei akutem Nierenversagen auftreten kann. Histonstimulation im Tubulus führte zur Rekrutierung von primären Immunzellen aus dem Endothel (C1) bis nach C3.
Zusammenfassung: Komplexe, 3-dimensionale und perfundierte Zellkulturmodelle erlauben durch Kokultivierung verschiedener Zelltypen die Abbildung funktionell wichtiger Abschnitte der Niere in vitro. Auf Basis dieser Arbeit könnte die Etablierung vielfältiger (Schädigungs-)Modelle zur besseren Abbildung und Untersuchung von in-vivo-Phänomenen möglich sein.

 
14-3-3 proteins stabilize KIBRA
P059 

F. Westphal, D. O. Wennmann, J. Kremerskothen, T. Weide, H. Pavenstädt; Münster

Objective: The scaffolding protein KIBRA is crucial for the cellular function of podocyts, which form a central element of the blood-urine filtration barrier in the kidney. Recent findings reveal that KIBRA plays an important role in the pathogenesis of podocyte injury by upstream regulation of the Hippo pathway. Other studies demonstrate that KIBRA proteins regulate the motility of podocytes. The family of 14-3-3 proteins has over fivehundred identified ligands, and are known to regulate enzymatic activity, subcellular localization, stabilizing multiprotein complexes, and blocking other interaction sites of their target proteins. Here, we analize the interaction of 14-3-3 proteins with KIBRA.
Method: Co-immunoprecipitation of KIBRA (FLAG-(M2)- or GFP-tagged) and 14-3-3 proteins was performed with transfected HEK293T cells. Western blot analysis with the displayed specific antibodies included a phospho-specific antibody to the binding motif at Threonin 929 of KIBRA. Threonin 929 was exchanged by Alanin to create the non-phosphorylatable mutant KIBRA T929A. Furthermore, a C- (including T929) and a N-terminal mutant of KIBRA were created. Stable, doxycycline-inducible HEK293T cell lines with KIBRA and KIBRA T929A were generated by lentiviral gene transduction using the pINDUCER21 system. The signal intensity of western blots was quantificated by the software ImageJ. The calculation of the decay constants with 99 % confidence intervals was performed by students t test. All calculations and exponential regressions were done using GraphPad prism.
Results: Co-immunoprecipitation experiments confirmed the interaction of KIBRA with the 14-3-3 isoforms ε, η, β, τ and γ. The interaction of WWC2 and WWC3 with 14-3-3 τ and ε could be shown in a similar experiment. The phospho-specific antibody binds only to a phosphorylated KIBRA T929 site. Figure 1 E shows that 14-3-3 proteins bind only to KIBRA mutants, which contain the required phosphorylated T929 binding site.

P059-1


Next, we treated pairs of induced pInducer KIBRA WT and T929A cell lines with cycloheximide to inhibit further protein synthesis, and compared the protein decay. Figure 2 reveals that KIBRA T929A, which is unable of binding to 14-3-3 proteins, decays significantly faster than KIBRA WT.

P059-2


Conclusion: We conclude that 14-3-3 proteins interact with the phosphorylated KIBRA T929 binding site and this interaction stabilizes KIBRA. These results suggest that 14-3-3 proteins regulate the activity of the scaffolding protein KIBRA.

 
The Transcriptional Regulator Tfap2a in Renal Collecting Duct Barrier Formation
P060 

J. Leiz, C. Hinze, K. M. Schmidt-Ott; Berlin

Objective: To ensure urinary concentration is strictly controlled and to avoid paracellular diffusion, the renal collecting duct is comprised of a tight epithelial barrier resulting in a strict separation of intraluminal urine and the interstitium. Disruption of this barrier leads to impaired water reabsorption and loss of adequate urine concentration ability. The overall transcriptional network controlling the collecting duct barrier formation is only incompletely characterized. Using an integrated bioinformatics approach, we identified Tfap2a, a factor implicated in epithelial differentiation, as a candidate transcriptional regulator that might be involved in the transcriptional control of this process.
Method: Inner medullary collecting duct (IMCD3) cells were engineered to harbour CRISPR/Cas9-induced knockouts (KO) of Tfap2a. Additionally, a collecting duct-specific knockout of Tfap2a in mice was generated. Deregulated genes were identified by mRNA sequencing and the in vivo model was used in metabolic studies to analyse urinary concentration ability.
Results: mRNA sequencing and subsequent gene ontology analysis revealed a strong impact of Tfap2a on the expression of important junctional components. For example, Ocln and Tjp2 were downregulated in both models when compared to WT controls. In addition, Tfap2a-KO mice showed a slightly reduced urine osmolality and altered urinary electrolyte levels under baseline conditions in comparison to control littermates indicating a mild defect in urinary concentration.
Conclusion: Our data support the hypothesis that Tfap2a might be involved in the transcriptional regulation of barrier formation in renal collecting ducts. A detailed understanding of the underlying network mechanisms might provide important insights into the potential involvement in kidney disease.

 
A novel analysis workflow for single nephron GFR measurement via intravital two-photon microscopy in mice.
P061 

F. Kessel, H. Kröger, J. Sradnick, M. Gerlach, V. Todorov, C. Hugo; Dresden

Objective: Intravital microscopy in animal models is an emerging technique in life sciences with advanced applications in kidney research. In particular, the measurement of single nephron (SN) GFR in mice comprises a method to assess a key parameter of kidney function. After intravenous injection of a freely filtered fluorescent dye a time series is captured and the filtration in single glomeruli is measured by two-photon laser scanning microsopy. Filtration is observed from the intraglomerular capillaries to the connected proximal tubulus (PT) and the intratubular dye intensity shift is measured from the transition from the glomerular urinary pole to the connected PT. However, existing methods for the analysis of the image data in rats (Kang et al. 2006) had limited robustness in mice. Due to the reduction in size, higher curvature and therefore smaller distances for the aquisition along the PT, the results were highly variable depending on the position of the manually set measurement points. Moreover the total tubular volume was solely estimated based on length and diameter in 2D images.
Method: We extended the published workflow in "ImageJ" by continuous rather than punctual measurement of fluorescence intensity along the PT in the time series. Additional modelling of the actual tubular volume in a 3D dataset replaced the inaccurate estimation of the tubular volume, to increase robustness, accuracy and objectivity. Subsequent data analysis in "R" normalized the shift of signal position along the PT over time against the tubular volume to calculate the filtrated volume per second by linear regression.

P061


Results: With the previously published method the results were highly variable. After repeated analysis of the same image material (10 different glomeruli in 5 animals, analyzed 5 times by the same researcher), the finally calculated GFR varied by a mean relative standard deviation of 41%. By reducing the user interaction and data interpretation, with our method, the variation could be reduced to 14%. When applying this analysis to the image data aquired in healthy and diabetic C57BL/6J mice, we could detect an increase of 4 fold SN GFR in diabetic mice when compared to the healthy mice.
Conclusion: To increase the reliabilty of SN GFR measurements by intravital microscopy in mice, we extended an existing workflow by continuous measurement, 3D-modelling and sophisticated data analysis while reducing manual interaction. Application to microscopy data acquired in diabetic and healthy mice prove the general applicability, high reliability and clinical relevance of this novel analysis approach.

 
Centrosomal protein 83 (CEP83) is required for kidney progenitor differentiation from human induced pluripotent stem cells(hiPSCs)
P062 

F. Mansour, K. M. Schmidt-Ott; Berlin

Objective: Centrosomal protein 83 (CEP83) is a component of the distal appendages (DAPs) of centrioles, which is necessary for the assembly of primary cilia (Tanos et al.,2013). Previous studies have implicated primary cilia in normal development and tissue homeostasis, including kidney development (Hildebrandt et al.,2011). The kidney develops from hiPSCs in a stepwise process involving induction of specialized progenitors in the intermediate mesoderm, followed by their differentiation into kidney epithelia. CEP83 mutations have been linked with human nephronophthisis (Failler et al., 2014). Here, we used a human induced pluripotent stem cell (hiPSCs) derived system that models the induction of intermediate mesoderm (IMM) progenitors followed by their kidney epithelial differentiation (Takasato et al., 2016) to analyze the role of CEP83 in human kidney epithelial differentiation.
Method: We used CRISPR-CAS9 technology to induce inactivating deletions of CEP83 in human iPSCs. We then used a stepwise induction protocol to differentiate intermediate mesoderm cells in a monolayer culture followed by an organoid system to induce nephrons. We used morphological analyses, gene expression studies, and immunostaining to analyze intermediate mesoderm and nephron differentiation.
Results: Wildtype iPSCs successfully differentiated into kidney organoids containing differentiated nephron epithelia in 67% (N=99), which was associated with an up-regulation of key nephron markers, including PAX8, SLC12A1, NPHS1, and PODXL. In contrast, CEP83-mutated cells failed to differentiate into nephron epithelia (N=104). Mechanistic studies indicated increased numbers of proliferating and apoptotic cells in CEP83-deficient IMM and an enhanced induction of odd-Skipped Related Transcription Factor 1 (OSR1)-positive intermediate mesoderm. In contrast, genes indicative of nephron progenitor differentiation (e. g. JAG1) and nephron differentiation (PAX8, SLC12A1, NPHS1, and PODXL) were downregulated in CEP-83-deficient vs. wildtype organoids.
Conclusion: In a human system modeling kidney epithelial differentiation from hiPSCs, CEP83 was required for the induction of nephron epithelia. Our data provide insight into the role of CEP83 in cellular differentiation and may help to better understand the pathogenesis of CEP83-associated renal developmental defects.

 
RORyt+Foxp3+ biTregs are broadly immunosuppressive effector Tregs and functionally differ from Th17 specific Treg17 cells
P063 

G. R. Herrnstadt, C. Niehus, J. Hagenstein, T. Ramcke, A. Nosko, M. Warkotsch, M. N. Wong, V. Puelles, T. B. Huber, M. A. Kluger, O. M. Steinmetz; Hamburg

Objective: Recently, Tregs expressing the Treg master transcription factor Foxp3, together with the Th17 charateristic RORyt have been identified (biTregs). Due to technical limitations, the effect of complete absence of biTregs, as opposed to conditional knockout of RORyt in Tregs, is unknown to date. Furthermore, it is currently unclear, whether biTregs preferentially suppress Th2 or Th17 responses. Finally, whether and how biTregs differ from Th17 specific CCR6+ RORγt- Treg17 cells remains controversial.
Method: FACSorted CD4+ T cells from fluorescence reporter mice, containing or lacking wild type or CCR6-/- biTregs, were transferred into RAG1-/- mice and NTN glomerulonephritis was studied. Immune responses were analysed after transfer of exogenous biTregs and in mice with Treg selective RORγt deficiency. Intrarenal interactions of biTregs with other leukocyte populations were analysed by immunofluorescence. In vitro suppression assays of biTregs versus Treg17 cells were performed. The role of CCR6 on biTregs for trafficking and homeostasis was analysed in vivo and in vitro.
Results: Mice lacking biTregs showed aggravated NTN. Treg selective RORγt deficiency but not lack of biTregs resulted in uncontrolled Th2 immunity. Importantly, Th17 responses also remained unchanged. In line, exogenous transfer of biTregs had broad immunosuppressive effects, with no preference for Th2 or Th17 responses. Furthermore, high-resolution tissue analyses of renal biTregs showed no preferential co-localization with Th17 cells. While CCR6+RORγt- Treg17 cells effectively suppressed IL-17 production in vitro, addition of biTregs even enhanced IL-17 secretion. Moreover, biTreg restricted lack of CCR6 did not alter Th17 immunity but nevertheless resulted in aggravation of NTN. Interestingly, renal trafficking was not relevantly impaired by CCR6 deficiency. Rather, we found that CCR6+ biTregs had a highly activated phenotype and impaired CCR6 signaling resulted in reduced biTreg expansion. Likewise, treatment of Tregs with the specific CCR6 ligand CCL20 augmented biTreg generation.
Conclusion: Our new model for the first time allows to study effects of complete absence of biTregs. biTregs showed broad immunosuppressive effects in glomerulonephritis, with no preference for downregulation of Th2 or Th17 responses. Interestingly, while Treg17 cells use the CCR6 for spatial co-localization and suppression of Th17 cells, biTregs require CCR6 for homeostasis and activation. In summary our studies identify biTregs as potent effector Tregs which are functionally different from Treg17 cells.

 
Circular RNA-based biomarker profile of Fabry Disease
P064 

A. Nowak, G. Haddad, A. Kistler, S. Nlandu-Khodo, F. Beuschlein, R. P. Wüthrich, J. Lorenzen, M. Kölling; Zürich/CH, Frauenfeld/CH

Objective: Fabry disease is a rare X-linked lysosomal storage disease caused by mutations in the galactosidase α gene. Deficient activity of α-galactosidase A leads to glycosphingolipid accumulations in multiple organs. Circular RNAs represent strong regulators of gene expression. Their circular structure ensures high stability in blood. We hypothesized that blood-based circular RNA profiles improve phenotypic assignment and therapeutic monitoring of Fabry Disease.
Method: A genome-wide circular RNA expression analysis was performed in blood of genetically diagnosed patients with Fabry Disease (n=58), age- and sex matched healthy volunteers (n=14) and disease control patients with acute kidney injury (n=109). Most highly dysregulated circular RNAs were validated by quantitative real-time polymerase chain reaction. Linear regression analyses were conducted for validated circular RNA biomarkers and clinical patient characteristics.
Results: A distinct circular RNA transcriptome signature identified patients with Fabry Disease. Circular RNAs hsa_circ_0006853, hsa_circ_0083766 and hsa_circ_0002397 distinguished patients with Fabry Disease from both healthy controls and patients with acute kidney injury. Hsa_circ_0002397 demonstrated, furthermore, a female-specific circular RNA expression pattern. Circular RNA level were significantly related to galactosidase α gene mutations, early symptoms, phenotypes, disease severities, specific therapies and long-term complications of Fabry Disease.
Conclusion: The discovery of circular RNA-based and Fabry Disease-specific biomarker may advance future diagnosis and therapeutic monitoring to diminish long-term complications of Fabry Disease.

 
mTOR-Inhibition als potentieller Rescue-Mechanismus für Podozyten mit akkumulierten DNA-Schäden
P065 

L. Blomberg, F. Braun, B. Schumacher, B. Schermer, T. Benzing, C. Kurschat; Köln, Hamburg

Objective: Chronic kidney disease is a major problem in aging societies. Unfortunately, adequate models to study kidney aging are lacking. In a previous study, we were able to show that a podocyte-specific knockout of the DNA damage repair gene Ercc1 leads to podocyte aging and the development of FSGS in a murine model. We have shown that mTOR is significantly upregulated in Ercc1-deficient mouse podocytes. In this study we have analyzed the effect of mTOR inhibition in this aging podocyte mouse model.
Method: Ercc1flox/flox mice were bred in a mixed CD1/FVB background and crossed with CD1 mice expressing Cre recombinase under the podocin promoter. At 4 and 8 weeks, mice were treated with rapamycin or set on a caloric restriction regimen. At 8 and 12 weeks of age weight, urine and serum were analyzed. Kidneys were fixed in paraformaldehyde and embedded in paraffin, fresh-frozen or prepared for immunohistochemistry.
Results: Ercc1pko mice treated with rapamycin at the age of 8 weeks showed no significant beneficial effect for survival or improved kidney function in comparison to control mice. Starting treatment at an earlier timepoint (4 wks) lead to a reduction of mTOR activity in glomeruli of rapamycin-treated female mice. Female Ercc1pko mice on caloric restriction showed reduced proteinuria in comparison to control mice.
Conclusion: Our study underlines the need to further investigate the role of podocyte DNA damage and therapeutic interventions to inhibit the mTOR pathway. We showed that caloric restriction has a protective effect on proteinuria in a mouse model developing chronic kidney disease due to DNA damage accumulation.

 
The Dual Endothelin/Angiotensin II Receptor (ETAR/AT1R) Antagonist Sparsentan Slows Renal Disease, Improves Lifespan, and Attenuates Hearing Loss in Alport Mice: Comparison with Losartan and Atrasentan
P066 

D. Cosgrove, B. Dufek, D. Delimont, D. Meehan, G. Samuelson, J. Hartsock, G. Phillips, R. Gill, J. Hasson, C. Jenkinson, R. Komers, M. A. Gratton; Omaha/USA, St. Louis/USA, San Diego/USA

Objective: In Alport syndrome (AS), ET A R activation is important in renal and inner ear pathologies. Previously, we showed that sparsentan (SP) prevented increases in proteinuria, fibrosis, glomerulosclerosis, and glomerular basement membrane dysmorphology in AS mice. Here we compare the effect of SP, the AT1 R antagonist losartan (LS), and the ETA R antagonist atrasentan (ATR) on lifespan and proteinuria in AS mice treated from 4 weeks (W), and the effect of SP and LS on hearing loss and inner ear pathology in mice treated from 3 W to 8.75 W.
Method: Wild type (WT) and AS mice were orally gavaged daily with vehicle (V), 60 or 120 mg / kg of SP (SP60, SP120), or LS (20 mg / kg; 3-4 W) or were given LS ( 10 mg / kg from 4 W) or ATR (7.5 mg / kg [in females] or 10 mg / kg [in males]) in the drinking water. Two studies were conducted: early intervention for hearing from 3-8.75 W (V, SP120, and LS, n = 5) and for lifespan with treatment from 3 W (V, n = 10) or from 4 W (SP60, SP120, LS, or ATR n = 5). Urinary protein / creatinine ratio (UP / C) was assessed weekly. The auditory brainstem response (ABR) was used to assess hearing ability and sensitivity to noise at 8 (pre-noise) -8.75 (post-noise) W. The cochleae were preserved and strial pathology was determined by transmission electron microscopy.
Results: SP120 significantly (P <0.05) increased median lifespan compared to any other group (Figure 1) (median lifespan [days]; 67.5 V; 84 LS; 74.5 ATR; 81 SP60; 118 SP120). At 8 W, only SP120 significantly (P <0.05) attenuated the increase in UP / C compared to V (UP / C mg / mg mean ± SD: 47 ± 16 V; 31 ± 6 LS; 42 ± 18 ATR; 61 ± 44 SP60; 20 ± 3 SP120). UP / C at 11 W in SP120 mice was significantly attenuated (P <0.05) compared to that in LS mice. SP120 significantly improved post-noise ABR thresholds at 16 kHz compared to V mice (* P <0.05), LS was without effect (Figure 2). Furthermore, dysmorphology of the stria vascularis was noted in LS but not SP120-treated AS mice.

P066


Conclusion: SP120 in AS mice significantly extended lifespan beyond that of mice treated with SP60, LS, or ATR and attenuated noise-induced hearing loss compared to V while LS had no effect. Sparsentan may therefore offer a novel, dual-therapeutic approach in AS by reducing both renal injury and hearing loss.

 
Die FERM Domäne von Protein 4.1O interagiert mit Polycystin-1
LA13 

T. E. Vienken, H. Ruffing, E. Königshausen, L. C. Rump, L. Sellin; Düsseldorf

Hintergrund: Beim Großteil der ADPKD-Patienten liegt eine Mutation im PKD1 vor. PKD1 kodiert für Polycystin-1. Für die Zystogenese wird eine Störung der Proliferation der Tubuluszellen beschrieben. Protein 4.1O ist ein Adapterprotein des Aktinzytoskeletts, welches Membranrezeptoren mit dem Aktinzytoskelett verbindet. Gleichzeitig sind für Protein 4.1O Eigenschaften eines Tumorsuppressorgens (Oncogene, 2007) bekannt. Bisher sind für PKD1 keine Interaktionen mit Aktinadaptorproteinen und dem Aktinzytoskelett bekannt. 
Methode: Mittels Überexpression wurden PKD1-Fulllength und Verkürzungsmutanten transient exprimiert. Aus den Zelllysaten wurde Polycystin-1 immobilisiert und mittels Western Blot die Interaktion mit Protein 4.1O und seinen Verkürzungsmutanten untersucht. Neben Verkürzungsmutanten, die N- und C-Terminal den Einfluss der Protein 4.1O Domänen Band 4.1, FERM, Aktin-Bindungsdomäne und der C-terminalen Domäne untersucht haben, wurde auch eine coiled coil Deletionsmutante von Protein 4.1O hergestellt. Pulldownexperimente mit bakteriell rekombinantem cytoplasmatischem PKD1 und HEK293T-Lysaten wurden durchgeführt.
Ergebnisse: Coimmunipräzipitationen zeigen eine Interaktion von Protein 4.1O mit Polycystin-1 fulllength.
Der C-Terminus von Polycystin-1 interagiert mit allen 3 Isoformen von Protein 4.1O (201, 204, 207). Die Deletion der coiled coil Domäne in Protein 4.1O verhindert die Interaktion zum C-Terminus von Polycystin-1 nicht. Die N-terminalen Anteile von Protein 4.1O, die die F1 und F2-lobes der FERM-Domäne beinhalten, sind ausreichend für die Interaktion mit den Polycystin-1 C-Terminus.
Zusammenfassung: Die FERM-Domäne von Protein 4.1O interagiert mit dem C-Terminus von Polycystin-1. Die Interaktion von Protein 4.1O ist der erste Linker von Polycystin-1 zum Aktin-Zytoskelett. Die Auswirkung der Protein-Protein-Interaktion auf die Kanalaktivität, die Proliferation und Aktivität vom Transkriptionsfaktor c-myc sind aktuell Gegenstand weiterer Untersuchungen. Darüberhinaus muss die funktionelle Konsequenz dieser Interaktion in Tiermodellen für ADPKD untersucht werden.

 
Charakterisierung eines isoform-spezifischen Protein 4.1O-Antikörpers
LA14 

H. Ruffing, T. E. Vienken, E. Königshausen, L. C. Rump, L. Sellin; Düsseldorf

Hintergrund: Protein 4.1O ist ein Aktinadaptorprotein, welches in einem GWAS für die diabetische Nephropathie bei Diabetikern (Typ 1) als ein Kandidatengen beschrieben wurde (Pezzolesi,MG 2009). Die dort identifizierten SNPs mappen in die Promotorregion von FRMD3, welches für Protein 4.1O kodiert. Protein 4.1O ist ein Mitglied der FERM-Domänen Proteine mit einer N-Terminalen FERM-Domäne, eine Aktinbindungsdomäne und einem c-Terminus, welcher u.a. an Membranrezeptoren binden kann (z.B. Nephrin). Interessanter Weise gibt es im Homo sapiens und nicht in Mus musculus von Protein 4.1O mehrere Isoformen mit unterschiedlichen N- und C-terminalen Anteilen, welche in der Lage sind, die Bindungen der FERM-Domäne und zu den Membranrezeptoren zu beeinflussen.
Methode: Es wurde ein isoformspezifischer Antikörper gegen die C-terminale Langversion des Protein 4.1O mittels Immunisierung gegen ein bakteriell rekombinantes Antigen der letzten C-terminalen 54 Aminosäuren von Protein 4.1O in Huhn und Kaninchen erzeugt. Die Antiseren wurden Affinitätsaufgereinigt und nachfolgend an rekombinanten Proteinen und Zelllysaten validiert. Darüberhinaus wurden mit den rekombinanten Proteinfragmenten von Protein 4.1O Pulldown-Experimente mit eukaryontischen Zelllysaten durchgeführt und mittels Western Blot Bindungspartner nachgewiesen.
Ergebnisse: Beide Immunisierungen - von Huhn und Kaninchen - waren primär erfolgreich. Das bakteriell rekombinante Antigen wird von den Antiseren vor und nach der Affinitätsaufreinigung erkannt. Im Westernblot kann das Antiserum zwischen den Protein 4.1O Isoformen 201/207 und 204 sicher unterscheiden. Im Pulldown mit bakteriell rekombinanten Proteinfragmenten von Protein 4.1O erkennen die Antiseren die Antigene und die Proteinfragmente binden den C-Terminus von eukaryontischem Nephrin.
Zusammenfassung: Der isoformspezifisch Antikörper gegen Protein 4.1O differenziert zwischen den unterschiedlichen C-terminalen Isoformen im Westernblot aus Zelllysaten und im Pulldown zwischen unterschiedlichen bakteriell rekombinanten Fusionsproteinen des Proteins 4.1O. Die weiteren Anwendungen in der Immunfluoreszenz, Immunhistochemie und Immunpräzipitation sind Gegenstand aktueller Versuche.

 
YB-1 mediates TNFR-induced pro-survival signaling by regulating NF-kB activation
LA15 

A. Shah, C. Plaza-Sirvent, S. Weinert, J. Buchbinder, I. Lavrik, P. R. Mertens, I. Schmitz, J. Lindquist; Magdeburg, Bochum

Objective: Cell fate decisions regulating survival and death are essential for maintaining tissue homeostasis; dysregulation thereof can lead to tumor development. In some cases, survival and death are triggered by the same receptor, e.g. tumor necrosis factor (TNF)-receptor 1 (TNFR1).
Method: To investigate the contribution of YB-1 to TNF receptor signaling, we utilize wild type and Ybx1-deficient macrophages, as well as control and YBX1 knockdown cell lines. NF-κB activation was visualized using imaging flow cytrometry and western blotting. The influence on cell survival was determined by flow cytometry.
Results: We identified a prominent role for the cold shock Y-box binding protein-1 (YB-1) in the TNF-induced activation and nuclear translocation of NF-κB p65. In the absence of YB-1, the expression of TNF receptor-associated factor 2 (TRAF2), a central component of the TNF receptor signaling complex required for NF-κB activation, is significantly reduced. Therefore, we hypothesize that the loss of YB-1 results in a destabilization of TRAF2. Consistent with this hypothesis, we observe that YB-1-deficient cells are more prone to TNF-induced apoptotic cell death. We observe enhanced effector caspases 3 activation and can successfully rescue the cells using the pan-caspase inhibitor zVAD-fmk, but not necrostatin-1.
Conclusion: Taken together, our results indicate that YB-1 plays a central role in promoting cell survival and identifies a novel mechanism by which enhanced YB-1 expression may contribute to tumor development.

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P062

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P063

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