Der sGC-Aktivator Cinaciguat als Therapieoption der diabetischen Nephropathie und Untersuchung von glukoseinduzierten molekularen Mechanismen in Mesangialzellen

Die Anzahl der Patienten, die an Diabetes mellitus erkranken und als Langzeitfolge eine diabetische Nephropathie (DN) entwickeln, steigt stetig. Weltweit stellt die DN die Hauptursache der terminalen Niereninsuffizienz dar, die im ungünstigsten Fall eine Dialy-se-Therapie oder Nierentransplantation notwendig macht. Bis heute existieren immer noch keine zielgerichteten Behandlungsmöglichkeiten für die DN. Als Standardtherapie erhalten die Patienten im Allgemeinen Inhibitoren des Renin-Angiotensin-Aldosteron-Systems, welche den Progress der Erkrankung zwar verlangsamen aber nicht zu einer Heilung füh-ren. In verschiedenen Studien konnte bereits gezeigt werden, dass die Aktivierung der NO/sGC/cGMP-Signalkaskade durch sGC-Aktivatoren effektiv diabetesinduzierte Nieren-schädigungen verhindern konnte. Aus diesem Grund wurde in dieser Arbeit untersucht, ob der sGC-Aktivator Cinaciguat einen protektiven Einfluss auf die Entwicklung der DN in einem murinen Typ-1-Diabetesmodell hat. Hierfür wurden sowohl diabetische Wildtyp- als auch eNOS-KO-Mäuse in einem Zeitraum von acht oder zwölf Wochen untersucht. Die Hälfte der Tiere erhielt in den letzten vier Wochen des jeweiligen Versuchszeitraums zudem Cinaciguat über das Futter verabreicht. Insbesondere eNOS-KO-Mäuse nach zwölf Wochen Diabetes entwickelten deutliche Merkmale einer humanen DN, wie eine reduzierte glomeruläre Filtrationsrate, erhöhte Se-rum-Kreatininspiegel, Albuminurie, Expansion des Mesangiums und Nierenfibrose. In Tie-ren, die zusätzlich Cinaciguat erhalten haben, konnte diese Nierenschädigung jedoch er-folgreich reduziert werden. Um außerdem einen detaillierteren Einblick in die zugrundeliegenden molekularen Mecha-nismen zu erhalten, wurde zusätzlich die Expression verschiedener Signalproteine der sGC/cGMP/PKG-Signalkaskade im Nierengewebe aber auch in primären Mesangialzellen analysiert. Erstmals wurde beispielsweise die Expression der Phosphodiesterase (PDE) 9a in der diabetischen Niere analysiert, wobei eine signifikante Reduktion dieses Enzyms festgestellt werden konnte. Auch Thrombospondin 1 (TSP1) wurde genauer untersucht. Als Regulator des Zytokins TGFβ, nimmt TSP1 eine Schlüsselfunktion in fibrotischen Nie-renerkrankungen ein. Sowohl in den diabetischen Nieren als auch in den mit Glukose sti-mulierten Mesangialzellen konnten deutlich erhöhte TSP1-Level beobachtet werden. Die pharmakologische Aktivierung der sGC/cGMP/PKG-Signalkaskade mit Cinaciguat oder 8-Br-cGMP konnte diese Expressionszunahme jedoch zuverlässig hemmen. Durch weitere Zellkulturversuche mit PKG-KO-MCs wurde schließlich untersucht, ob die regulatorischen Effekte auf die TSP1-Expression eventuell durch die PKGs vermittelt wer-den. In diesem Zusammenhang wurde zum ersten Mal überhaupt nachgewiesen, dass neben der PKGI auch die PKGII in Mesangialzellen exprimiert wird. Da die Reduktion der TSP1 mit Cinaciguat und 8-Br-cGMP jedoch sowohl in PKGI-KO als auch in PKGII-KO-Zellen weiterhin detektierbar war, wurde postuliert, dass möglicherweise beide Isoformen der PKG in Kombination an der Regulation der TSP1 beteiligt sind. Zusammengefasst konnte in dieser Dissertation gezeigt werden, dass die pharmakologische Aktivierung der NO/sGC/PKG-Signalkaskade mit Cinaciguat einen protektiven Effekt auf die Niere unter hyperglykämischen Bedingungen hat. sGC-Aktivatoren stellen somit grundsätzlich eine vielversprechende Therapieoption für die DN dar. Des Weiteren konnten neue Einblicke in zugrundeliegenden molekularen Mechanismen gewonnen werden, die dazu beitragen, die diabetische Nierenerkrankung besser zu verstehen

Establishing a Split Luciferase Assay for Proteinkinase G (PKG) Interaction Studies

Nitric oxide (NO/cyclic guanosine monophosphate (cGMP)-regulated cellular mechanisms are involved in a variety of (patho-) physiological processes. One of the main effector molecules in this system, proteinkinase G (PKG), serves as a molecular switch by phosphorylating different target proteins and thereby turning them on or off. To date, only a few interaction partners of PKG have been described although the identification of protein-protein interactions (PPI) is indispensable for the understanding of cellular processes and diseases. Conventionally used methods to detect PPIs exhibit several disadvantages, e.g., co-immunoprecipitations, which depend on suitable high-affinity antibodies. Therefore, we established a cell-based protein-fragment complementation assay (PCA) for the identification of PKG target proteins. Here, a reporter protein (click beetle luciferase) is split into two fragments and fused to two different possible interaction partners. If interaction occurs, the reporter protein is functionally complemented and the catalyzed reaction can then be quantitatively measured. By using this technique, we confirmed the regulator of G-Protein signaling 2 (RGS2) as an interaction partner of PKGI alpha (a PKG-isoform) following stimulation with 8-Br-cGMP and 8-pCPT-cGMP. Hence, our results support the conclusion that the established approach could serve as a novel tool for the rapid, easy and cost-efficient detection of novel PKG target proteins

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Activation of soluble guanylyl cyclase signalling with cinaciguat improves impaired kidney function in diabetic mice

Background and Purpose Diabetic nephropathy is the leading cause for end-stage renal disease worldwide. Until now, there is no specific therapy available. Standard treatment with inhibitors of the renin-angiotensin system just slows down progression. However, targeting the NO/sGC/cGMP pathway using sGC activators does prevent kidney damage. Thus, we investigated if the sGC activator cinaciguat was beneficial in a mouse model of diabetic nephropathy, and we analysed how mesangial cells (MCs) were affected by related conditions in cell culture. Experimental Approach Type 1 diabetes was induced with streptozotocin in wild-type and endothelial NOS knockout (eNOS KO) mice for 8 or 12 weeks.. Half of these mice received cinaciguat in their chow for the last 4 weeks. Kidneys from the diabetic mice were analysed with histochemical assays and by RT-PCR and western blotting. . Additionally, primary murine MCs under diabetic conditions were stimulated with 8-Br-cGMP or cinaciguat to activate the sGC/cGMP pathway. Key Results The diabetic eNOS KO mice developed most characteristics of diabetic nephropathy, most marked at 12 weeks. Treatment with cinaciguat markedly improved GFR, serum creatinine, mesangial expansion and kidney fibrosis in these animals. We determined expression levels of related signalling proteins. Thrombospondin 1, a key mediator in kidney diseases, was strongly up-regulated under diabetic conditions and this increase was suppressed by activation of sGC/cGMP signalling. Conclusion and Implications Activation of the NO/sGC/PKG pathway with cinaciguat was beneficial in a model of diabetic nephropathy. Activators of sGC might be an appropriate therapy option in patients with Type 1 diabetes

Targeted Delivery of Soluble Guanylate Cyclase (sGC) Activator Cinaciguat to Renal Mesangial Cells via Virus-Mimetic Nanoparticles Potentiates Anti-Fibrotic Effects by cGMP-Mediated Suppression of the TGF-β Pathway

Diabetic nephropathy (DN) ranks among the most detrimental long-term effects of diabetes, affecting more than 30% of all patients. Within the diseased kidney, intraglomerular mesangial cells play a key role in facilitating the pro-fibrotic turnover of extracellular matrix components and a progredient glomerular hyperproliferation. These pathological effects are in part caused by an impaired functionality of soluble guanylate cyclase (sGC) and a consequentially reduced synthesis of anti-fibrotic messenger 3′,5′-cyclic guanosine monophosphate (cGMP). Bay 58-2667 (cinaciguat) is able to re-activate defective sGC; however, the drug suffers from poor bioavailability and its systemic administration is linked to adverse events such as severe hypotension, which can hamper the therapeutic effect. In this study, cinaciguat was therefore efficiently encapsulated into virus-mimetic nanoparticles (NPs) that are able to specifically target renal mesangial cells and therefore increase the intracellular drug accumulation. NP-assisted drug delivery thereby increased in vitro potency of cinaciguat-induced sGC stabilization and activation, as well as the related downstream signaling 4- to 5-fold. Additionally, administration of drug-loaded NPs provided a considerable suppression of the non-canonical transforming growth factor β (TGF-β) signaling pathway and the resulting pro-fibrotic remodeling by 50–100%, making the system a promising tool for a more refined therapy of DN and other related kidney pathologie

Inflammatory fibroid polyp: A series of 29 cases and a systematic review of the literature

An inflammatory fibroid polyp (IFP) of the gastrointestinal tract is a localized, benign mesenchymal lesion consisting of spindle-shaped stromal cells, eosinophilic granulocytes, and some lymphocytes and plasma cells. The discovery of a frequent mutation of the platelet-derived growth factor receptor A (PDGFRA) gene was the first hint of a gene-regulating process in IFPs. The aim of this study was to investigate the interaction of inflammatory processes and the role of mutation and expression of the PDGFRA gene in the development of IFPs for the first time. We used immunohistochemistry to analyze the composition of inflammatory cells and next generation sequencing (NGS) to provide a broad overview of gene mutations.We report on 29 cases of IFP. The mean age, gender differences, and localization were compatible with the literature. Spindle cell histomorphology was present in 79% of cases showing a typical onion skin-like perivascular arrangement and significantly high CD34 positivity (p = 0.002, Fisher’s exact test). Eosinophilic granulocytes were present in an average density of 60 ± 49/high power field (HPF) (range: 15–200), and there was a significantly higher rate of IFPs larger than 2 cm in size (p = 0.018, Wilcoxon test). All but one cases could be analyzed by NGS. Mutations were observed in 17 cases (60.7%), including 13 (46.4%) mutations in the PDGFRA gene. Among the gastric lesions, mutations were found in exon 18 of the PDGFRA gene with amino acid exchange (Asp842Val) for eight out of 10 cases and in exon 12 in two cases. All three cases in the small intestine revealed mutation of the PDGFRA gene in exon 12. We found no PDGFRA mutation in our colonic cases. PDGFRA expression was significantly correlated with mutations of the same gene (p = 0.005, Fisher’s exact test) and especially with mutations in exon 12 of the same gene (p < 0.001, Fisher’s exact test). Interestingly, three of our cases (10.3%) without mutation or expression of the PDGFRA gene revealed an unusually high concentration of IgG-positive plasma cells (average: 140 ± 26/HPF, range: 110–160) and IgG4-positive plasma cells (average: 87 ± 21/HPF, range: 60–100). For comparison, an IgG4/IgG ratio of more than 0.4 is commonly observed in IgG4-related diseases. Our molecular results were in accordance with 113 genetically analyzed cases published to date. There was a correlation between the IFP site and mutation variants of the PDGFRA gene. IFPs were localized in the stomach in 49.1% of cases, in the small intestine in 47.3%, and in the colon in 3.6%. Exon 12 of the PDGFRA gene was mutated in 41.1% of cases and primarily occurred in the small intestine (82.6%). Exon 18 was mutated in 22.3% of cases and primarily occurred in the stomach (80.0%). The mutated codon interval 566–571 in exon 12 and codon 842 in exon 18 were compatible, as observed in a gastrointestinal stromal tumor. Conclusively, the correlation between mutation and expression of the PDGFRA gene points to different pathways in IFPs. Additionally, our data hint at a morphological but not genetic overlap between IFPs and IgG4-related pseudotumors