Studies on the expression of the anti-inflammatory protein annexin A1 in the kidney

Persistierende Entzündungsreaktionen sowie die Akkumulation von fibrotischem Narbengewebe gelten als typische histopathologische Merkmale bei chronischen Nierenerkrankungen. Das antifibrotische, antiinflammatorische und Resolution- fördernde Protein Annexin A1 wird in der Niere kräftig exprimiert. Die Funktion und Regulation von Annexin A1 während der Entwicklung chronischer Nierenerkrankungen wurden jedoch bisher nicht systematisch untersucht. Ziel dieser Arbeit war daher die Untersuchung der renalen Annexin A1-Expression in der gesunden Niere sowie bei akuten und chronischen Nierenerkrankungen. Hierzu wurden die akute anti-Thy-1 Nephritis als inflammatorisches und transgene Ratten mit einer Überexpression von humanem Renin und humanem Angiotensinogen (dTGR) als chronisch fibrotisches Tiermodell gewählt. Die morphologische Analyse der renalen Parenchymschädigung erfolgte anhand von PAS-gefärbten Gewebeschnitten. Annexin A1-exprimierende Zellen im renalen Interstitium wurden durch immunhistochemische Doppelmarkierungen für Annexin A1 und etablierten Markern für die interstitiellen Zellen identifiziert. In den Tieren mit der anti-Thy-1 Nephritis erfolgte zudem die Bestimmung der Annexin A1-mRNA mittels quantitativer TaqMan Real-Time Polymerase Kettenreaktion. In den dTGR erfolgte eine Lokalisation Kollagen 1-exprimierender Fibroblasten mittels mRNA in-situ-Hybridisierungen. Die Ergebnisse der Arbeit zeigten eine kräftige Annexin A1-Expression im gesunden renalen Tubulointerstitium. Diese wurde zum größten Teil von Fibroblasten und Makrophagen getragen. Endothelzellen der größeren Gefäße wiesen ebenfalls eine starke Annexin A1-Expression auf. Im Gegensatz dazu zeigten die Endothelzellen von Arteriolen, Kapillaren und venösen Gefäße kein Annexin A1-Signal. Im Verlauf der anti-Thy-1 Nephritis fand sich initial eine rapide Mesangiolyse und tubulointerstitielle Inflammation, gefolgt von reparativen Prozessen und einer Rückbildung der Entzündungsreaktion (Resolution). Parallel fand sich ein rapider Anstieg der Expression von Annexin A1-mRNA und von Annexin A1-immunreaktiven Zellen im Interstitium der kranken Tiere. Lokalisationsstudien an Tag 5 und an Tag 15 nach Induktion der Nephritis zeigten eine zunehmende Anzahl Annexin A1-exprimierender interstitiellen Makrophagen. Hiermit konnte erstmalig demonstriert werden, dass interstitielle Makrophagen einen quantitativ relevanten Beitrag zur gesteigerten Annexin A1-Expression in der Ausheilungsphase einer renalen Inflammation leisten. Auch in den dTGR konnte eine Zunahme der interstitiellen Annexin A1-Expression nachgewiesen werden. Die Zunahme dieser Expression wurde hier primär durch alpha smooth muscle actin-negative, ruhende Fibroblasten getragen. Alpha smooth muscle actin-positive Myofibroblasten hingegen zeigten keine Annexin A1-Expression. Die gesteigerte Annexin A1-Expression im Rahmen einer chronischen fibrotischen Nierenerkrankung geht somit am ehesten von ruhenden Fibroblasten aus. Die Resultate dieser Arbeit zeigen, dass die Expression von Annexin A1 im renalen Interstitium bei Nierenerkrankungen mit unterschiedlicher Genese stimuliert wird. Hierzu tragen Makrophagen und Fibroblasten maßgeblich bei. Diese Ergebnisse stellen eine Basis für die Erforschung neuer antiinflammatorischer sowie Resolutions-fördernder Therapien bei der Behandlung von Nierenerkrankungen dar.Unresolved inflammation and accumulation of fibrotic scar tissue present major causes for the development of renal failure throughout the world. The intrinsic anti-fibrotic, anti-inflammatory and resolution-promoting protein Annexin A1 is abundantly expressed in the kidney but it´s role during the development of renal disease has not been elucidated. Aim of this study was to investigate the renal expression of Annexin A1 in animal models for acute and chronic kidney diseases. Anti-Thy-1 nephritic rats and hypertensive rats with an ubiquitous overexpression of the human genes for renin and angiotensinogen (dTGR) were used as animal models for inflammatory and fibrotic kidney disease, respectively. Morphological studies were performed using PAS-stained kidney sections. Localization studies were performed using double-labeling immunofluorescence for Annexin A1 and established markers for renal interstitial cells. In animals with anti-Thy-1 nephritis Annexin A1 mRNA abundance was determined by TaqMan real-time polymerase chain reaction. mRNA in-situ-hybridisation was used to investigate the expression of collagen 1 in dTGR. The results of this study demonstrated abundant expression of Annexin A1 in the interstitium of the healthy rodent kidney. Fibroblasts and macrophages were the principal Annexin A1 expressing cells at this site. Endothelial cells of the larger arterial vessels stained positive for Annexin A1, whereas those of the renal microvasculature and veins did not. Animals with anti-Thy-1 nephritis displayed rapid mesangiolysis and tubulointerstitial inflammation followed by reparative processes. mRNA for Annexin A1 and the number of macrophages were increased in the course of the nephritis. Increased Annexin A1 expression was detected in macrophages at d5 and, more pronounced, at d15. This study thus showed for the first time that macrophages significantly contribute to the increased abundance of Annexin A1 during the resolution phase of nephritic disease. An increased interstitial abundance for Annexin A1 protein was also detected in the kidneys of dTGR. Here, Annexin A1 signal was primarily localized to alpha smooth muscle actin negative dormant fibroblasts whereas alpha smooth muscle actin positive myofibroblasts did not express Annexin A1. These findings indicate that the increased expression of Annexin A1 in renal fibrosis is attributable to the renal fibroblasts. The results of this study allow the conclusion that the expression of Annexin A1 is stimulated during the course of kidney disease. In this context macrophages and fibroblasts play a major role. This knowledge will serve as a foundation for the investigation of novel anti-inflammatory and pro-resolving pathways during renal diseases

Immunosuppressive effects of circulating bile acids in human endotoxemia and septic shock: patients with liver failure are at risk

Abstract Background Sepsis-induced immunosuppression is a frequent cause of opportunistic infections and death in critically ill patients. A better understanding of the underlying mechanisms is needed to develop targeted therapies. Circulating bile acids with immunosuppressive effects were recently identified in critically ill patients. These bile acids activate the monocyte G-protein coupled receptor TGR5, thereby inducing profound innate immune dysfunction. Whether these mechanisms contribute to immunosuppression and disease severity in sepsis is unknown. The aim of this study was to determine if immunosuppressive bile acids are present in endotoxemia and septic shock and, if so, which patients are particularly at risk. Methods To induce experimental endotoxemia in humans, ten healthy volunteers received 2 ng/kg E. coli lipopolysaccharide (LPS). Circulating bile acids were profiled before and after LPS administration. Furthermore, 48 patients with early (shock onset within  0.4 μg/kg/min) and 48 healthy age- and sex-matched controls were analyzed for circulating bile acids. To screen for immunosuppressive effects of circulating bile acids, the capability to induce TGR5 activation was computed for each individual bile acid profile by a recently published formula. Results Although experimental endotoxemia as well as septic shock led to significant increases in total bile acids compared to controls, this increase was mild in most cases. By contrast, there was a marked and significant increase in circulating bile acids in septic shock patients with severe liver failure compared to healthy controls (61.8 µmol/L vs. 2.8 µmol/L, p = 0.0016). Circulating bile acids in these patients were capable to induce immunosuppression, as indicated by a significant increase in TGR5 activation by circulating bile acids (20.4% in severe liver failure vs. 2.8% in healthy controls, p = 0.0139). Conclusions Circulating bile acids capable of inducing immunosuppression are present in septic shock patients with severe liver failure. Future studies should examine whether modulation of bile acid metabolism can improve the clinical course and outcome of sepsis in these patients. Graphical abstrac