Freitag, 02.10.2020

14:00 - 15:30

Poster DGfN 2020

Experimentelle Nephrologie 2 (P067 - P075; LA16 - LA18)

 
Erhöhte Expression von Glukose-Transportern bei Peritonealdialyse-Patienten
P067 

S. Schricker, T. Oberacker, M. Schanz, M. D. Alscher, M. Ketteler; Stuttgart

Hintergrund: Die Peritonealmembran wird chronisch durch Dialysate auf Glukosebasis belastet. Studien an Ratten und an menschlichen Primärzellen zeigen eine erhöhte Expression der Glukose-Transporter bei Exposition gegenüber Peritonealdialyse (PD)-Lösungen. Ziel dieser Studie war es, die Expression von Glukose-Transportern in humanen Peritonealbiopsien zu untersuchen.
Methode: Humanes Peritonealgewebe von gesunden Kontrollen, PD-Patienten sowie Patienten mit encapsulierender Peritonealsklerose (EPS) wurde auf GLUT1, GLUT3 und SGLT2 mittels mRNA-Expressionsanalysen (qPCR), die Proteinexpression der Transporter immunhistochemisch mit Hilfe eines Histo-Scores untersucht.
Ergebnisse: Insgesamt wurden Biopsien aus dem Peritoneum von 7 gesunden Kontrollen, 35 Patienten mit PD ohne Anzeichen von EPS sowie von 12 Patienten mit EPS analysiert. Die Kontrollgruppe war älter als die Patientenkohorte (Kontrolle: Median 64 Jahre, IQR: 53-70; PD: Median 60 Jahre, IQR: 46-69 und EPS: Median 51 Jahre, IQR: 45,25-58,75). Darüber hinaus waren mehr männliche Teilnehmer in der Kontrollgruppe (75%) als in der PD- (42%) und EPS-Gruppe (25%). Weiter war die PD-Dauer bei EPS-Patienten länger (Median 103,8 Monate) als bei PD-Patienten (Median 34,3 Monate).
mRNA-Expressionsanalysen zeigten eine deutlich steigende Tendenz für GLUT1 in EPS-Patienten im Vergleich zur Kontrollgruppe. Die Expression von GLUT3 und SGLT2 hingegen unterschied sich nicht signifikant. Auf Proteinebene ergibt sich lediglich für GLUT1 und SGLT2 eine erhöhte Expression in den PD-Gruppen. Während sich ein erhöhter Histo-Score für GLUT1 bei EPS-Patienten im Vergleich zu PD-Patienten ergibt, zeigt sich insbesondere für SGLT2 ein Unterschied bei EPS-Patienten im Vergleich zur Kontrollgruppe.

P067


Zusammenfassung: Diese Studie zeigt zum ersten Mal eine deutliche Tendenz zur Steigerung der GLUT1 und SGLT2 Expression im menschlichen Peritonealgewebe nach Exposition gegenüber PD-Lösungen, insbesondere bei EPS-Patienten. Des Weiteren zeigt diese Studie, dass SGLT2 ein vielversprechender therapeutischer Angriffspunkt zur Verbesserung der Peritonealmembranfunktion sein könnte.

 
Uremic Apelin and leucocytic Angiotensin Converting Enzyme as modulators of pro-atherosclerotic events in CKD Patients
P068 

B. Trojanowicz, C. Ulrich, M. Girndt; Halle (Saale)

Objective: Apelin peptides (APLN) serve as secondary substrates for Angiotensin Converting Enzyme 2 (ACE2) and in contrast to Angiotensin II (AngII), exert blood-pressure lowering and vasodilatation effects through binding to G-coupled APLN receptor (APLNR). ACE2-mediated cleavage of the APLN may decline its vasodilatory effects. On the other hand, ACE2 decrease may potentiate the hypotensive properties of APLN.
This study investigated the correlation between serum-APLN and leucocytic APLNR and ACE2 expression in patients with chronic kidney disease as well as the impact of APLN peptides on monocytic expression of ACE2 and behaviour of the monocytes under uremic conditions in vitro.
Method: Total serum APLN was measured with competitive ELISA assay. Expression of leucocytic ACE2, corresponding receptors and organic anion transporter SLCO2B1 was investigated in 24 CKD3-5 (74.5 ±10.7 years), 66 HD patients (63 ±14.7 years) and 32 healthy control subjects (NP, 53.1 ±6.8 years). THP-1 monocytes were treated with APLN -12, -13 and -36 peptides, co-treated with indoxyl sulphate, p-cresol and p-cresyl sulphate or pooled normal and uremic sera and investigated for APLNR, TNFa, IL-6 as well as ACE, ACE2 and ACE2-targeting miR-421. The functional effects of APLN peptides were investigated with transmigration and adhesion assays.
Results: The levels of serum APLN and leucocytic APLNR or SLCO2B1 expression were significantly elevated in uremic patients as compared with healthy individuals, and correlated with each other. Increased serum APLN correlated significantly with decreased ACE2 on uremic leucocytes. THP-1 monocytes treated with APLN peptides revealed significant increase of APLNR and ACE2, and reduction of pro-inflammatory TNFa and IL-6. Uremic toxins led to dramatic increase of miR-421 followed by significant reduction of ACE2 transcripts. Uraemia-mediated decrease in monocytic ACE2 could be partially re-induced with APLN peptides, especially APLN-13 and -36. Functional assays revealed that out of all APLN peptides tested, APLN-36 triggered the most potent transmigration and reduction of endothelial-adhesion as further demonstrated by diminished levels of MCSF.
Conclusion: Uremic patients show enhanced expression of total serum APLN and leucocytic APLNR, but decreased ACE2. Although APLN peptides may partly protect the decay of monocytic ACE2, uremic milieu is the most dominant modulator of local ACE2 and likely to contribute to the progression of atherosclerosis.

 
Structural analysis of the Crumbs polarity complex at the base of motile and non-motile cilia.
P069 

J. H. Hinrichs, H. E. G. Klapproth, P. I. Nedvetsky, M. Krahn; Münster

Objective: Ciliopathies are heterogenic genetic disorders that are caused by the dysfunction of motile or non-motile (primary) cilia, microtubule-based structures that protrude from various mammalian cells. Motile cilia can be found, inter alia, in the upper and lower respiratory tract where they are essential for the mucociliary clearance. Non-motile, primary cilia are apical membrane protrusions serving as sensory organelles and are involved in numerous cellular processes. Defective cilia are linked to several diseases, such as Primary Ciliary Dyskinesia (PCD) and Polycystic Kidney Disease (PKD). However, the underlying pathomechanisms are still poorly understood. Recently, evidence emerged that perturbation in cell polarity involving the Crumbs (CRB) polarity complex results in ciliary defects. The CRB complex consists of the transmembrane protein Crb3, its intracellular adaptor protein Pals-1 and the scaffolding protein PATJ. Ciliary proteins have been shown to interact with components of the CRB complex and mice with knockouts of CRB complex proteins develop PKD or respiratory problems. Thus, we studied the role of CRB complex proteins in the morphogenesis of motile and non-motile cilia.
Method: In vitro experiments were performed using Madin-Darby Canine Kidney II (MDCK) cells cultured in both conventional and three-dimensional cell culture. For in vivo analysis whole-mount stainings of tissue from wild type CL57BL/6 mice were analyzed using confocal imaging.
Results: In addition to its localization at the cell-cell contacts, a Crb complex protein PATJ has been found both at the motile cilia in the mouse trachea and at the primary cilia in MDCK cells. Additionally, in MDCK cells, Pals1 and Crb3b were present at the primary cilium. CRISPR/Cas9 knock out of PATJ in MDCK cells resulted in dissociation of Pals-1 and Crb3 from primary cilia and defects in the cilia maintenance.
Conclusion: In this study, we identified CRB polarity proteins at motile and non-motile cilia and showed novel functions of the CRB complex proteins in the maintenance of primary cilia. Our data underline the importance of cell polarity for the correct function of primary cilia and its potential role in the pathomechanism of ciliopathies such as PCD and PKD.

 
Bioinformatische Betrachtung von aberrant filtrierten Proteinen beim nephrotischen Syndrom anhand der GO- und KEGG-Datenbanken
P070 

M. Wörn, K. Boldt, M. Ueffing, F. Artunc; Tübingen

Hintergrund: Beim nephrotischen Syndrom (NS) kommt es zu einer aberranten Filtration von großmolekularen Plasmaproteinen in den Urin. Über einen Vergleich der Proteine im nephrotischen Human- und Mausurin mithilfe der den Proteinen zugeordneten GO- (Gene Ontology) und KEGG (Kyoto Encyclopedia of Genes and Genomes)-Bezeichnungen ist noch wenig bekannt. Dies könnte wichtige Hinweise über mögliche pathophysiologische Vorgänge wie bei der Natriumretention und der Ödementstehung liefern.

Methode: Die Urinproteine wurden aus Patienten mit akutem NS (n=10, Proteinurie 7,8±1,2 g/g Kreatinin) und Mäusen mit Doxorubicin-induziertem NS (n=6, 167±39 g/g Kreatinin) im Vergleich zu Proben von gesunden Personen und Mäusen mittels Tandem-Massenspektrometrie identifiziert. Die im nephrotischen Urin signifikant neu auftretenden Proteine wurden mithilfe der GO- und KEGG-Datenbanken bioinformatisch analysiert.

Ergebnisse: Im nephrotischen Urin von Patienten und Mäusen wurden n=64 bzw. n=83 differenziell vorkommende Proteine identifiziert. Im Humanurin waren unter der GO-Kategorie „Biologischer Prozess“ Proteine mit der GO-Bezeichnung „Akute Entzündungsreaktion“ (n=22) am stärksten vertreten, gefolgt von „Protein-Aktivierungskaskade“ (n=19), „Thrombozyten-Degranulation“ (n=18) und „Komplementaktivierung“ (n=15, Irrtumswahrscheinlichkeit = FDR jeweils <10-20). Unter der GO-Kategorie „Zelluläre Komponente“ dominierten die GO-Bezeichnungen „Blutplasma“ und „extrazellulär“ (FDR jeweils <10-20). Unter der GO-Kategorie „Molekulare Funktion“ fanden sich Proteine mit den GO-Bezeichnungen „Protease-Inhibition“ (n=19), sowie „Serinproteasen“ (n=7) als dominante Proteaseklasse, am häufigsten (FDR jeweils <10-5). In der KEGG-Datenbank-Analyse waren diese Proteine hauptsächlich dem Komplement- und Gerinnungssystem zuzuordnen. Stark angereichte Proteine waren u.a. Plasminogen, Faktor VII-aktivierende Protease (FSAP) sowie verschiedene Komplementfaktoren. Bei den nephrotischen Mausproben zeigt sich in allen Kategorien ein sehr ähnliches Muster.

Zusammenfassung: Im nephrotischen Urin von Patienten und Mäusen finden sich hauptsächlich Plasmaproteine, die dem Komplement- und Gerinnungssystem zuzuordnen sind. Sie könnten eine wichtige Rolle im pathophysiologischen Ablauf des NS spielen, was durch gezielte Experimente, z.B. mit Knockout-Mäusen, validiert werden muss.

 
Generation of anti-THSD7A antibodies using a human antibody phage display library
P071 

S. Köllner, L. Seifert, P. A. Heine, M. Hust, N. M. Tomas; Hamburg, Braunschweig

Objective: Membranous nephropathy (MN) is an antibody-mediated autoimmune disease. In the majority of cases, autoantibodies target the podocyte membrane proteins PLA2R1 and THSD7A. THSD7A consists of 21 TSP-1 domains (d1 to d21). We could previously demonstrate that autoantibodies in THSD7A-associated MN cause disease and recognize several extracellular domains of the target antigen. However, it remains unclear how this antibody polyreactivity relates to disease pathogenicity and whether antibody elimination represents an effective therapeutic strategy. The aim of this study was the generation of anti-THSD7A antibodies using a human antibody phage display library for further pathomechanistic and therapeutic investigations.
Method: Phage display links phenotype and genotype by connecting the genetic information for pIII-coupled single chain fragment variables (scFv) contained in the phagemid to the proteins displayed on the phage particle surface. It is a round-based method to select high affinity scFvs binding to any antigen presented to the library. In this case, a naïve library with a diversity of 1.5x1010 was used to identify binders to the extracellular part of human and mouse THSD7A. Binders were sequenced, cloned into scFv-Fc format, and produced in HEK293-6E cells. Binding of scFv-Fc to human and mouse THSD7A was investigated using Western blot, a d1_d21 ELISA, and indirect immunofluorescence testing (IFT). Epitope regions were further defined using an ELISA in which always two to three domains were coated.
Results: Four positive binders were selected (B1-B4). While B1 and B3 only interact with human THSD7A, the other scFv-Fc bind human and mouse THSD7A in IFT. In Western blot, B1 exclusively recognized human THSD7A while B2-B4 recognized mouse and human THSD7A. B1 also recognized human d1_d21 in ELISA and human d6_d7 in the domain-specific ELISA. B2 recognized both human and mouse d1_d21 in ELISA and mouse d1_d2 in the domain-specific ELISA. B3 did not recognize human or mouse d1_d21 in ELISA, but human and mouse d1_d2 in the domain-specific ELISA. B4 did not react with d1_d21 in ELISA nor with any domain in the domain-specific ELISA.
Conclusion: Phage display represents a powerful method to generate antigen-specific antibodies. The antibodies selected here show different binding characteristics in vitro. In vivo binding characteristics and pathogenic potential need to be determined. The scFv-Fc can represent a starting point for the evaluation of innovative therapeutic strategies such as the elimination of THSD7A-specific autoantibodies to reduce podocyte injury in MN.

 
Die Rolle von NIPP1 bei Hypoxie in den Podozyten
P072 

M. Liebisch, G. B. Wolf; Jena

Hintergrund: Hypoxie induziert Mechanismen, welche die Proteinurie begünstigen und wesentlich zur Progression diabetischer Nierenerkrankungen beitragen. Dabei sind die genauen Mechanismen noch weitestgehend unbekannt. Eine entscheidende Rolle spielt HIF-1a (hypoxia inducible factor). Studien zeigten, dass HIF-1a in den Podozyten die Expression von Proteinen des Schlitzdiaphragma unterdrückt und damit die Auslöschung der Füßchenfortsätze fördert. In der Literatur ist beschrieben, dass eine Korrelation in der Expression von HIF-1a und NIPP1 (nuclear inhibitor of proteinphosphatase 1) existiert. Untersuchungen unserer Arbeitsgruppe zeigten, dass die Expression von NIPP1 in den Podozyten unter diabetischen Bedingungen vermindert ist. Zudem verdeutlichten unsere Analysen, dass NIPP1 in den Podozyten an der EZH2 (enhancer of zeste homolog)-vermittelten repressiven Trimethylierung von H3K27 beteiligt ist und die Suppression von NIPP1 die Expression zahlreicher Schädigungs-relevanter Moleküle in den Podozyten induziert, die charakteristisch für diabetische Nierenerkrankungen sind. Das Ziel dieser Untersuchungen war, die Rolle von NIPP1 bei Hypoxie in den Podozyten zu analysieren.
Methode: Murine, differenzierte Podozyten wurden für 24 Stunden bei 1% O2 inkubiert bzw. für 24 Stunden mit 100 µM CoCl2 inkubiert. Um die Expression von NIPP1 in den Podozyten zu unterdrücken, wurden die Zellen für 24 Stunden mit 20 nM NIPP1 siRNA inkubiert. Die mRNA-Expression wurde mit Real-Time PCR untersucht, die Proteinexpression wurde über Immunfluoreszenzanalysen visualisiert.
Ergebnisse: Die Analysen zeigten, dass sowohl hypoxische Bedingungen (1% O2) als auch die Stabilisierung von HIF-1a durch Behandlung der Zellen mit CoCl2 die Expression von NIPP1 in den Podozyten signifikant vermindert. Des Weiteren belegten unsere Untersuchungen, dass die Suppression von NIPP1 die podozytäre Expression von HIF-1a beeinflusst.
Zusammenfassung: Neben dem bereits bekannten Beitrag der Suppression von NIPP1 an pathophysiologischen Vorgängen in den Podozyten, wie einer zellulären Hypertrophie, spielt die verminderte Expression von NIPP1 auch bei hypoxischen Prozessen eine entscheidende Rolle.

 
Transcriptional mechanisms of renal tubule cell specification in reprogramming and CAKUT
P073 

K. Grand, M. Stolz, G. Salinas, M. Getwan, R. Pichler, S. Lienkamp; Zürich/CH, Freiburg, Göttingen

Objective: Transcription factors that control early renal development have been found to be the protein products of genes frequently mutated in human congenital renal malformations. HNF1B is one of these and involved in a number of signaling pathways and plays a significant role in both nephrogenesis and maintenance of renal tubular functions. HNF1B is also essential for direct reprogramming of fibroblasts to murine induced renal tubule epithelial cells (iREC) and can induce ectopic pronephric tissue in Xenopus animal caps. Some mutations of HNF1B found in patients with cystic-dysplastic kidneys are still able to contribute to both reprogramming and ectopic renal tissue induction.The aim of this project is to find potential direct and indirect targets of HNF1B and characterize the functional consequences of patient-specific HNF1B mutations in Xenopus.
Method: We used iRECs and the renal-like tissue induced from Xenopus animal caps to investigate patient-specific HNF1B mutations causing developmental renal abnormalities. Transcriptional changes altered by HNF1B mutations were characterized in both of the kidney models with bulk RNA analysis. To study conserved transcriptional mechanisms of HNF1B transcriptome analysis from two different species was combined with in vivo experiments.
Results: Preliminary results from the transcriptomic analysis of iRECs and animal caps identified how the global transcriptional profile of iRECs is altered by one mutation (R295C) in HNF1B and shows striking differences in signaling pathways associated with renal morphogenesis and organic anion transport. Targeting hnf1b in Xenopus embryos with CRISPR / Cas9 method or morpholino oligomer showed a phenotype of delayed or disrupted kidney development.
Conclusion: In conclusion, the combined use of directly reprogrammed mammalian cells and Xenopus experiments allow us to gain a unique perspective into evolutionarily conserved mechanisms of renal development and human disease.

 
Detection of new human splice forms of renin
P074 

O. Malecha, S. Reuter, R. Mrowka; Jena

Objective: The Renin-angiotensin-aldosterone system (RAAS) is significant for blood pressure regulation and fluid-electrolyte homeostasis. Renin, an aspartyl-protease expressed in juxtaglomerular cells of the kidney, is the key enzyme of RAAS. Malfunction of RAAS might lead to hypertension and cardiovascular diseases; therefore, understanding of molecular mechanisms of renin expression is significant.
A few transcript variants of renin that result in different protein isoforms have been described in the literature so far. The nucleotide sequence of renin splice variant REN-201 in human has ten coding exons, a transcript length of 1,462 base pairs (bps) and consist of 406 amino acid residues. Splice variant REN-202 includes nine coding exons out of twelve exons; it has a transcript length of 1,488 bps and contains 368 amino acid residues.
Here we describe three new alternative splicing variants of the human renin gene.
Method: Human embryonic kidney 293 cell line (HEK293) was obtained from the DSMZ-German collection of microorganisms and cell cultures. A Cas9-dependent Synergistic Activation Mediator system was utilized for renin transcription activation. Next, total RNA was isolated, and complementary DNA was synthesized in a reaction catalyzed by the enzyme reverse transcriptase. Afterward, the polymerase chain reaction was performed to amplify the sequence of the transcript REN-201. The amplification products were visualized after performing the agarose gel electrophoresis, and the nucleotide order of DNA fragments was determined after DNA sequencing.
Results: We report the identification of three new alternative splicing variants of the human renin gene. The first splice form differs from the previously described forms as following: exons five, six, and seven were missing. In the second splice form, exons from five to nine were missing. The third splice form was missing the exon six. The missing exons encode a part of the aspartic peptidase, the active site of renin.
Conclusion: Different transcript variants of the renin gene may lead to the translation of the proteins with diverse biological functions. Here we described three new alternative splicing variants of the human renin gene, which differ in their amino acid sequence encoding for the aspartic peptidase, the active site of renin.

 
Identification of a novel putative candidate gene for ciliary chondrodysplasia and cystic kidney disease
P075 

M. Getwan, A. Hoppmann, P. Schlosser, K. Grand, W. Song, F. Heeg, S. Schroda, R. Diehl, E. Lausch, A. Köttgen, S. Lienkamp; Zürich/CH, Freiburg

Objective: Ciliary chondrodysplasia belongs to the big field of ciliopathies with skeletal malformations (shortened ribs and long bones, polydactyly) and a nephronophthesis-like phenotype (NPHP). Other organ manifestations include liver fibrosis, laterality defects and retinopathy. To shed light on the molecular pathogenesis of ciliary chondrodysplasia, its connection to NPHP and to expand our knowledge about the role of ciliary proteins during skeletal and renal development, we (1) established models of ciliary chondrodysplasia genes and (2) attempted to identify putative disease-related candidate genes.
Method: We established disease models for two ciliary chondrodysplasia genes (ift80, ift172) with CRISPR/Cas9 in Xenopus tropicalis. F0 animals were analyzed for their phenotype during tadpole and froglet stages. The functionality of the embryonic kidney was tested and ciliation analyzed by immunofluorescence. MicroCT-scans of froglets were performed to quantify limb abnormalities and kidney morphology. RNAseq of ift80 and ift172 LOF animals was performed to find changes in gene expression. (2) Novel disease-related genes were searched using an in silico screen, and candidates were evaluated according to disease criteria established in the models of disease genes (ift80 and ift172).
Results: (1) Loss of ift80 and ift172 resulted in typical phenotypes observed in ciliary chondrodysplasia patients. The functionality of embryonic kidneys was strongly impaired, and post-metamorphotic froglets developed cystic kidney disease. Cilia formation was reduced in mutant embryos. Froglets had additionally severe limb defects and polydactyly. RNAseq analysis revealed expression changes of genes interacting with the sonic hedgehog signaling pathway. (2) Based on the in silico screen, we examined four genes in more detail. One of these fulfilled all requirements to be a potential new ciliary chondrodysplasia gene (cilia defects, cystic kidneys, shortened limbs, polydactyly).
Conclusion: In conclusion, we established three models for (1) two already known and (2) one new ciliary chondrodysplasia and cystic kidney disease gene in Xenopus, which may provide new insights in the disease and molecular aspects of bone and kidney development.

 
Modelling the renoprotective role of prostaglandin reductase 2 (PTGR2) in zebrafish
LA16 

A. Kourpa, E. Mangelsen, J. Bolbrinker, R. Kreutz, D. Panakova; Berlin

Objective: Prostaglandins and particularly prostaglandin E2 (PGE2) play a crucial role in the initiation and progression of kidney inflammation and chronic kidney disease (CKD). Physiologically, PGE2 increases glomerular filtration barrier (GFB) permeability leading to proteinuria and its upregulation in podocytes is linked to increased fluid flow shear stress (FFSS) as observed during glomerular hyperfiltration. Our previous studies in Munich Wimstar Frömter (MWF) rat model suggest PTGR2 as a novel potential candidate for proteinuria development. PTGR2 is an important enzyme involved in the prostaglandin pathway as it terminally inactivates PGE2 signaling. Our aim is to establish zebrafish as a model to study the role of PTGR2 in kidney physiology and to investigate the mechanisms of how PTGR2 and particularly its substrate 15-keto-PGE2 contribute to the initiation and/or progression of CKD.
Method: The ptgr2 loss-of-function was studied using the morpholino (MO) knockdown technology. Upon the injection of ptgr2-ex2sd MO to the one-cell stage zebrafish embryos, the morphological changes of the pronephros and the proteinuria-like phenotype are observed between 48 hpf (hours post-fertilization) and 120 hpf of development. Using pharmacological approach, the embryos were exposed to different concentrations of the indomethacin (30uM-100uM), a well-characterized prostaglandin synthesis inhibitor, starting from 6hpf until 48hpf. The lipidomic analysis and the levels of eicosanoids were performed and analyzed by LIPIDOMIX GmbH.
Results: Our preliminary studies in zebrafish embryos demonstrate the loss of ptgr2 leads to a functional permeability defect in GFB, thus mimicking a proteinuria-like phenotype. In addition, reduced ptgr2 levels cause profound morphological changes to the zebrafish embryonic kidney, which appeared to be partially rescued after Indomethacin treatment. However, lipidomic analysis in the whole embryos (N=6, n=150) showed lower levels of PGE2 and 15-keto-PGE2 in the ptgr2 deficient embryos, arising thus the question of the presence of an alternative pathway that may regulates the levels of PGE2 in the absence of PTGR2.
Conclusion: Altogether, our data suggest a developmental role for PTGR2 in kidney morphogenesis and support the hypothesis that PTGR2 may function renoprotectively in CKD. Moreover, these findings establish the zebrafish as a valid model to study prostaglandin pathway in kidney morphogenesis and physiology, allowing us to study the PGE2/15-keto-PGE2/PTGR2 pathway as a potential target for renoprotection.

 
Hochauflösende Immunfluoreszenzmikroskopie durch Expansionsmikroscopy (ExM) von Schlitzmembranproteinen
LA17 

T. Schmitz, E. Königshausen, L. C. Rump, L. Sellin; Düsseldorf

Hintergrund: Die Darstellung des glomerulären Filters ist bisher die exklusive Domäne der Elektronenmikroskopie. Der apparative Aufwand hierfür ist enorm und die Freiheiten der Antikörpermarkierung sind gering. Die bisherigen lichtmikroskopischen Verfahren waren entweder durch eine zu geringe Auflösung (konventionelle Lichtmikroskopie) oder durch ebenfalls hohen technischen Aufwand (STORM, STED) limitiert.
Die Expansionsmikroskopie (ExM) ermöglicht, durch eine physikalische "Vorvergrößerung" der Probe deutlich höhere Auflösungen (bis zu 25 nm) mit einem Fluoreszenzmikroskop zu erzielen.   
Methode: Cos-Zellen, welche podozytäre Proteine (Podocin, Nephrin) exprimieren, werden fixiert und mittels Primär- und Sekundärantiköper bzw. Toxin fluoreszenzmarkiert. Die Zellen werden in ein Hydrogel eingebettet. Chemisch werden die Fluorophore mit dem Gel gekoppelt und nachfolgend die Zellen / extrazelluläre Matrix im Gel verdaut. Durch Immersion des hyperosmolaren Hydrogels in entionisiertem Wasser kommt es zu einer Expansion des Hydrogels, in welchem die an das Gel gekoppelten Fluorophore in gleicher Weise expandieren. Das expandierte Hydrogel wird in Kulturschalen mit geeignetem Glasboden in einem konventionellen Fluoreszenzmikroskop mikroskopiert. Der tatsächliche Expansionsfaktor wird mittels prä- und post Expansionaufnahmen ermittelt.
Ergebnisse: Auf dem ersten Bild

LA17-1

sind Phalloidin gefärbte Cos-Zellen vor der Expansion dargestellt. Auf dem zweiten Bild

LA17-2

sind die gleichen Zellen nach der Expansion abgebildet. Im post-Expansionsbild ist ein Doppelfärbung zu sehen. Beide Bilder sind mit dem gleichen 20x-Objektiv aufgenommen. Phalloidin ist rot und Podocin ist grün gefärbt. An der Zellmembran in vermutlichen fokal Adesions sieht man eine Kolokalisation von Podocin und Aktin (gelb). Der Vergrößerungsfaktor durch die Expansion beträgt ~4-fach.
Zusammenfassung: Die Expansionsmikroskopie ermöglicht eine hochauflösende Darstellung der Zielstrukturen mit einem konventionellen Fluoreszenzmikroskop. Eine besondere Sorgfalt ist bei der Auswahl der Fluorophore bedeutsam, da nicht alle Fluoreszenzfarbstoffe mit den chemischen Modifikationen der Probe kompatibel sind. Die hier gezeigte Technik lässt sich auch auf Gewebeschnitte übertragen. Es darf erwartet werden, dass mit der Expansionsmikroskopie, Multicolour-Labeling an der glomerulären Schlitzmembran mit Auflösungen von bis zu 25 nm im niedrigen ultrastrukturellen Bereich möglich wird.

 
Enhanced LPS-induced inflammation is dependent on intracellular YB-1
LA18 

L. Amann, S. Brandt, J. Schiwietz, P. R. Mertens; Magdeburg

Objective: Severe sepsis and septic shock are manifestations of bacterial infection in humans that are systemic with ensuing organ failure. The cold shock Y-box binding protein 1 (YB-1) is a mediator of both systemic and local inflammatory responses. Several inflammatory stimuli, including LPS, are known to induce an intracellular upregulation and secretion of YB-1. So far, the receptor Notch-3 is reported as the receptor for extracellular YB-1. Although YB-1 has been implicated in the regulation of pleiotropic cellular functions, the role of YB-1 in infectious diseases is largely unknown.
Method: In order to explore the role of YB-1 in infectious diseases, we have set up an LPS toxicity model with different animal strains: (i) wild type animals (C57BL/6), (ii) a tamoxifen-inducible whole body knockout (YB-1ΔRosaCreERT), (iii) a secretion-deficient murine model (YB-1K299A/301A knockin) crossed to RosaCreERT. Mice were challenged with LPS according to two distinct protocols, one with high doses (30mg/kg body weight) and acute toxicity, the other with low doses (1mg/kg LPS every 2nd day over 14 days) and chronic sublethal damage. We applied flow cytometry, cytokine and chemokine measurements using Bioplex, Western blot analysis, RT-qPCR, immunohistochemistry, and determined serum parameters (serum creatinine, ALAT, BUN, blood count, LDH). Furthermore, primary tubular cell cultures as well as bone marrow-derived macrophages were generated from the described strains to serve as model systems.
Results: While only 30% of wild type mice survived the LPS challenge, 75% of the YB-1 knockouts survived. The effect is more pronounced for the knockin mice with merely 5% survival 72h following LPS. This we link to changes in the inflammatory milieu which reveals an inverse correlation between cytokine levels (IL-6, IL-8, IL-12, IL-17, CCL5, Eotaxin, GCSF, MCSF, MCP-1, MIP1α, MIP1β) and survival. LPS-induced wild type and YB-1 knockin mice showed significant leukocytosis, whereas YB-1 knockout mice failed to do so. Furthermore, chronic administration of LPS resulted in progressive infiltration of immune cells within the kidneys at day 7 following LPS and is abrogated at day 14. Finally, our data link intracellular YB-1 concentrations to NF-?B pro-survival signaling.
Conclusion: Having observed enhanced survival in YB-1 knockouts and enhanced mortality in secretion-deficient mice we conclude that intracellular YB-1 orchestrates the pro-inflammatory milieu and hypothesize that extracellular YB-1 rather contributes to the resolution of inflammation.

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