Involvement of cyclic guanosine monophosphate-dependent protein kinase I in renal antifibrotic effects of serelaxin

Introduction: Kidney fibrosis has shown to be ameliorated through the involvement of cyclic guanosine monophosphate (cGMP) and its dependent protein kinase I (cGKI). Serelaxin, the recombinant form of human relaxin-II, increases cGMP levels and has shown beneficial effects on kidney function in acute heart failure patients. Antifibrotic properties of serelaxin are supposed to be mediated via relaxin family peptide receptor 1 and subsequently enhanced nitric oxide/ cGMP to inhibit transforming growth factor 1)) (TGFI)) signaling. This study examines the involvement of cGKI in the antifibrotic signaling of serelaxin. Methods and Results: Kidney fibrosis was induced by unilateral ureteral obstruction in wildtype (WT) and cGKI knock-out (KO) mice. After 7 days, renal antifibrotic effects of serelaxin were assessed. Serelaxin treatment for 7 days significantly increased cGMP in the kidney of WT and cGKI-KO. In WT, renal fibrosis was reduced through decreased accumulation of collagenl A1, total collagen, and fibronectin. The profibrotic connective tissue growth factor as well as myofibroblast differentiation were reduced and matrix metalloproteinases-2 and-9 were positively modulated after treatment. Moreover, Smad2 as well as extracellular signal-regulated kinase 1 (ERK1) phosphorylation were decreased, whereas phosphodiesterase (PDE) 5a phosphorylation was increased. However, these effects were not observed in cGKI-KO. Conclusion: Antifibrotic renal effects of serelaxin are mediated via cGMP/cGKI to inhibit Smad2- and ERK1-dependent TGF-13 signaling and increased PDE5a phosphorylation

Keratin Retraction and Desmoglein3 Internalization Independently Contribute to Autoantibody-Induced Cell Dissociation in Pemphigus Vulgaris

Pemphigus vulgaris (PV) is a potentially lethal autoimmune disease characterized by blister formation of the skin and mucous membranes and is caused by autoantibodies against desmoglein (Dsg) 1 and Dsg3. Dsg1 and Dsg3 are linked to keratin filaments in desmosomes, adhering junctions abundant in tissues exposed to high levels of mechanical stress. The binding of the autoantibodies leads to internalization of Dsg3 and a collapse of the keratin cytoskeleton-yet, the relevance and interdependence of these changes for loss of cell-cell adhesion and blistering is poorly understood. In live-cell imaging studies, loss of the keratin network at the cell periphery was detectable starting after 60 min of incubation with immunoglobulin G fractions of PV patients (PV-IgG). These rapid changes correlated with loss of cell-cell adhesion detected by dispase-based dissociation assays and were followed by a condensation of keratin filaments into thick bundles after several hours. Dsg3 internalization started at 90 min of PV-IgG treatment, thus following the early keratin changes. By inhibiting casein kinase 1 (CK-1), we provoked keratin alterations resembling the effects of PV-IgG. Although CK-1-induced loss of peripheral keratin network correlated with loss of cell cohesion and Dsg3 clustering in the membrane, it was not sufficient to trigger the internalization of Dsg3. However, additional incubation with PV-IgG was effective to promote Dsg3 loss at the membrane, indicating that Dsg3 internalization is independent from keratin alterations. Vice versa, inhibiting Dsg3 internalization did not prevent PV-IgG-induced keratin retraction and only partially rescued cell cohesion. Together, keratin changes appear very early after autoantibody binding and temporally overlap with loss of cell cohesion. These early alterations appear to be distinct from Dsg3 internalization, suggesting a crucial role for initial loss of cell cohesion in PV

cGMP becomes a drug target

Cyclic guanosine 3′,5′-monophosphate (cGMP) serves as a second messenger molecule, which regulates pleiotropic cellular functions in health and disease. cGMP is generated by particulate or soluble guanylyl cyclases upon stimulation with natriuretic peptides or nitric oxide, respectively. Furthermore, the cGMP concentration is modulated by cGMP-degrading phosphodiesterases. Several targets of cGMP are utilized to effect its various cellular functions. These effector molecules comprise cGMP-dependent protein kinases, ion channels, and phosphodiesterases. During the last decade, it emerged that cGMP is a novel drug target for the treatment of pulmonary and cardiovascular disorders. In this respect, several drugs were developed, which are now in clinical phase studies for, e.g., pulmonary hypertension or cardiovascular diseases. These new drugs act NO-independently with/without heme on soluble guanylyl cyclases or induce subtypes of particular guanylyl cyclases and thereby lead to new therapeutic concepts and horizons. In this regard, the fifth cGMP meeting held in June 2011 in Halle, Germany, comprised the new therapeutic challenges with the novel functional and structural concepts of cGMP generating and effector molecules. This report summarizes the new data on molecular mechanisms, (patho)physiological relevance, and therapeutic potentials of the cGMP signaling system that were presented at this meeting

Funktionelle Studie der NO/cGMP Signalkaskade in Niere und Thrombozyten

Im ersten Teil dieser Arbeit wurde die renale Funktion von cGKI unter physiologischen und pathophysiologischen Bedingungen untersucht. Sowohl in Fibroblasten des Interstitiums als auch in Mesangialzellen des Glomerulus wurde immunhistochemisch eine hohe cGKI-Expression detektiert. Die Rolle von cGMP/cGKI für die interstitielle Fibrose wurde mittels unilateraler Ureter Ligation (UUL) untersucht. Im cortikalen Interstitium wurden beide Isoformen schwach detektiert, während im medullären nur die α-Isoform auffallend stark immunhistochemisch nachweisbar war. Bereits dieses Expressionsmuster implizierte, dass cGKIα im Interstitium vermutlich mehr Funktionen ausübt als cGKIβ. Diese funktionelle Differenzierung der beiden Isoformen wurde mehrfach bestätigt: Sowohl auf mRNA-, als auch auf Protein-Ebene war die cGKIα-Expression stärker fibrosebedingt hoch reguliert als die der β-Isoform. Hierbei wurde immunhistochemisch gezeigt, dass die Expressionszunahme der α-Isoform primär im kortikalen Interstitium vorzufinden war. Zudem wurden die mRNA-Mengen der α-Isoform nach YC1- bzw. ISDN-Gabe signifikant verändert, dagegen blieben die der β-Isoform nahezu unbeeinträchtigt. Weiter zeigten die Untersuchungen der Rescue-Mäuse, dass der protektive Effekt von cGMP unter anderem über cGKI vermittelt wurde: Während ohne ISDN Behandlung der Fibrose-Verlauf in WT- und Rescue-Tieren annähernd gleich fortgeschritten war, blieb nach Verabreichung des NO-Donors der zuvor in WT detektierte protektive Effekt in den Rescues nahezu aus. Dies lässt die Annahme zu, dass die antifibrotische Wirkung von NO/cGMP durch cGKI vermittelt wird. Im zweiten Teil der Arbeit wurde die Rolle des NO/cGMP/cGKI Signalweges in Thrombozyten untersucht. Die Thrombozytenaktivierung dient einerseits dem Wundverschluss, begünstigt jedoch auch arteriosklerotische Vorgänge. Deshalb ist die Aufklärung der pharmakologischen Interventionsmöglichkeit von besonders hohem Interesse. Eine kurze Zusammenfassung der Ergebnisse soll noch einmal die Bedeutung von IRAG, einem Signalprotein der NO/cGMP Signalkaskade, für die Thrombozyten belegen: Besonders die NO/cGMP-abhängige Hemmung der Aggregation wird primär über IRAG vermittelt. Zudem wird die parallel ablaufende Sekretion beider Granulaarten - „alpha“ und „dense“ Granula - mittels NO/cGMP über IRAG inhibiert. Interessant war vor allem, die nach Aktivierung der Thrombozyten erhöhte Aggregabilität der IRAG-KO Thrombozyten im Vergleich zu den WT Thrombozyten. Die Plättchenadhäsion und -aktivierung sind initiale Ereignisse der Thrombusbildung, so dass deren Inhibition eine Möglichkeit bietet, unerwünschte thrombotische Ereignisse zu verhindern

Cyclic Nucleotide Signalling in Kidney Fibrosis

Kidney fibrosis is an important factor for the progression of kidney diseases, e.g., diabetes mellitus induced kidney failure, glomerulosclerosis and nephritis resulting in chronic kidney disease or end-stage renal disease. Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were implicated to suppress several of the above mentioned renal diseases. In this review article, identified effects and mechanisms of cGMP and cAMP regarding renal fibrosis are summarized. These mechanisms include several signalling pathways of nitric oxide/ANP/guanylyl cyclases/cGMP-dependent protein kinase and cAMP/Epac/adenylyl cyclases/cAMP-dependent protein kinase. Furthermore, diverse possible drugs activating these pathways are discussed. From these diverse mechanisms it is expected that new pharmacological treatments will evolve for the therapy or even prevention of kidney failure