Effects of DPP-4 Inhibitors on the Heart in a Rat Model of Uremic Cardiomyopathy

BACKGROUND: Uremic cardiomyopathy contributes substantially to mortality in chronic kidney disease (CKD) patients. Glucagon-like peptide-1 (GLP-1) may improve cardiac function, but is mainly degraded by dipeptidyl peptidase-4 (DPP-4). METHODOLOGY/PRINCIPAL FINDINGS: In a rat model of chronic renal failure, 5/6-nephrectomized [5/6N] rats were treated orally with DPP-4 inhibitors (linagliptin, sitagliptin, alogliptin) or placebo once daily for 4 days from 8 weeks after surgery, to identify the most appropriate treatment for cardiac dysfunction associated with CKD. Linagliptin showed no significant change in blood level AUC(0-∞) in 5/6N rats, but sitagliptin and alogliptin had significantly higher AUC(0-∞) values; 41% and 28% (p = 0.0001 and p = 0.0324), respectively. No correlation of markers of renal tubular and glomerular function with AUC was observed for linagliptin, which required no dose adjustment in uremic rats. Linagliptin 7 µmol/kg caused a 2-fold increase in GLP-1 (AUC 201.0 ng/l*h) in 5/6N rats compared with sham-treated rats (AUC 108.6 ng/l*h) (p = 0.01). The mRNA levels of heart tissue fibrosis markers were all significantly increased in 5/6N vs control rats and reduced/normalized by linagliptin. CONCLUSIONS/SIGNIFICANCE: DPP-4 inhibition increases plasma GLP-1 levels, particularly in uremia, and reduces expression of cardiac mRNA levels of matrix proteins and B-type natriuretic peptides (BNP). Linagliptin may offer a unique approach for treating uremic cardiomyopathy in CKD patients, with no need for dose-adjustment

25 years of endothelin research: the next generation

In the past three decades, endothelin and endothelin receptor antagonists have received great scientific and clinical interest, leading to the publication of more than 27,000 scientific articles since its discovery. The Thirteenth International Conference on Endothelin (ET-13) was held on September 8–11, 2013, at Tokyo Campus of the University of Tsukuba in Japan. Close to 300 scientists from 25 countries from around the world came to Tokyo to celebrate the anniversary of the discovery of the endothelin peptide discovered 25 years ago at the University of Tsukuba. This article summarizes some of the highlights of the conference, the anniversary celebration ceremony, and particularly the participation of next generation of endothelin researchers in endothelin science and the anniversary celebration. As a particular highlight, next generation endothelin researchers wrote a haiku (a traditional form of Japanese poetry originating from consisting of no more than three short verses and 27 on, or Japanese phonetic units) to describe the magic of endothelin science which they presented to the conference audience at the anniversary ceremony. The text of each haiku – both in its original language together with the English translation – is part of this article providing in an exemplary fashion how poetry can be bridged with science. Finally, we give an outlook towards the next 25 years of endothelin research

Renal Effects of the Novel Selective Adenosine A1 Receptor Blocker SLV329 in Experimental Liver Cirrhosis in Rats

Liver cirrhosis is often complicated by an impaired renal excretion of water and sodium. Diuretics tend to further deteriorate renal function. It is unknown whether chronic selective adenosine A1 receptor blockade, via inhibition of the hepatorenal reflex and the tubuloglomerular feedback, might exert diuretic and natriuretic effects without a reduction of the glomerular filtration rate. In healthy animals intravenous treatment with the novel A1 receptor antagonist SLV329 resulted in a strong dose-dependent diuretic (up to 3.4-fold) and natriuretic (up to 13.5-fold) effect without affecting creatinine clearance. Male Wistar rats with thioacetamide-induced liver cirrhosis received SLV329, vehicle or furosemide for 12 weeks. The creatinine clearance of cirrhotic animals decreased significantly (−36.5%, p<0.05), especially in those receiving furosemide (−41.9%, p<0.01). SLV329 was able to prevent this decline of creatinine clearance. Mortality was significantly lower in cirrhotic animals treated with SLV329 in comparison to animals treated with furosemide (17% vs. 54%, p<0.05). SLV329 did not relevantly influence the degree of liver fibrosis, kidney histology or expression of hepatic or renal adenosine receptors. In conclusion, chronic treatment with SLV329 prevented the decrease of creatinine clearance in a rat model of liver cirrhosis. Further studies will have to establish whether adenosine A1 receptor antagonists are clinically beneficial at different stages of liver cirrhosis

Effect of SLV329, an adenosine A1-receptor antagonist, on renal function in thioacetamide-induced rat model of liver cirrhosis.

1 Einleitung und Fragestellung 1 2 Literaturübersicht 3 2.1 Adenosin 3 2.1.1 Aufbau, Metabolismus und Vorkommen 3 2.1.2 Klassifizierung und Funktion der Adenosinrezeptoren 3 2.1.3 Adenosin und die Leber 5 2.1.4 Adenosin und die Niere 7 2.2 Das hepatorenale Syndrom 11 2.2.1 Die Leberzirrhose 11 2.2.2 Niereninsuffizienz 12 2.2.3 Nierenversagen bei Lebererkrankungen 13 2.2.4 Das hepatorenale Syndrom 13 2.3 Das Tiermodell 22 3 Material und Methoden 24 3.1 Tiere und Tierhaltung 24 3.2 Versuchsdesign 24 3.3 Materialien 26 3.3.1 Der Wirkstoff SLV329 26 3.3.2 Antikörper 27 3.4 Methoden 28 3.4.1 Nicht-invasive Blutdruckmessung 28 3.4.2 Untersuchung im Stoffwechselkäfig 28 3.4.3 SLV329-Serumspiegelbestimmung 30 3.4.4 Organentnahme 30 3.4.5 Histologische Methoden 30 3.4.6 Western-Blot 34 3.4.7 Statistik 37 4 Ergebnisse 38 4.1 Körpergewichte 38 4.2 Organgewichte und makroskopische Beurteilung der Organe 40 4.3 Futteraufnahme 41 4.4 Wasseraufnahme 42 4.5 Ergebnisse der Serumuntersuchungen 43 4.5.1 Serumparameter zu Beginn des Versuches 43 4.5.2 Serumparameter in der achten Woche des Versuches 44 4.5.3 Serumparameter in Woche 16 des Versuches 45 4.5.4 Ergebnisse der SLV329-Konzentrationsbestimmung 47 4.6 Ergebnisse der Urinuntersuchungen 48 4.6.1 Urinparameter zu Beginn des Versuches 48 4.6.2 Urinparameter in der achten Woche des Versuches 49 4.6.3 Urinparameter in Woche 16 des Versuches 50 4.7 Glomeruläre Filtrationsrate 51 4.8 Blutdruck 53 4.9 Herzfrequenz 54 4.10 Ergebnisse der histologischen Untersuchungen 55 4.10.1 Untersuchung der Leber 55 4.10.2 Untersuchung der Nieren 57 4.11 Ergebnisse der Western-Blot-Untersuchungen 60 4.11.1 Adenosin- Rezeptorexpression in der Leber 60 4.11.2 Adenosin-Rezeptorexpression in den Nieren 62 4.12 Mortalität 64 5 Diskussion 65 5.1 Diskussion der Methoden 67 5.2 Diskussion der Ergebnisse 73 5.3 Schlussfolgerung und Ausblick 82 6 Zusammenfassung 83 7 Summary 85 8 Literaturverzeichnis 87 9 Abkürzungsverzeichnis 103 10 Anhang 104 11 Publikationen 110 12 Danksagung 111 13 Selbständigkeitserklärung 112Hintergrund - Das hepatorenale Syndrom (HRS) ist primär eine funktionelle Niereninsuffizienz bei einer fortgeschrittenen Lebererkrankung. Die jährliche Inzidenz des HRS beträgt bei Patienten mit dekompensierter Zirrhose 8%. Häufig kommt es bei Zirrhotikern aufgrund der Aszitestherapie mit Diuretika zur Verschlechterung der Nierenfunktion und zur Entstehung von HRS. Adenosin-A1-Rezeptoren (A1R) sind an der Pathogenese des HRS über mehrere Mechanismen beteiligt. Die A1R-Aktivierung ist essenziell für die Auslösung des Adenosinakkumulation-bedingten hepatorenalen Reflexes, wirkt stark vasokonstriktiv an den kortikalen Nierengefäßen und steigert die Natrium- und Wasserresorption in der Niere. Die Hemmung der A1R führt zu einem Anstieg der Diurese ohne Minderung der glomerulären Filtrationsrate (GFR). Bei Herzinsuffizienz-Patienten wurde durch die kombinierte Therapie mit Furosemid und einen A1R-Antagonisten eine Steigerung der Harnausscheidung ohne Verminderung der GFR erreicht. Im Rahmen der vorgelegten Arbeit wurden die Auswirkungen des A1R-Antagonisten SLV329 auf die Nierenfunktion und auf die Diurese in der experimentell erzeugten Leberzirrhose der Ratte untersucht. Methoden – Für die Untersuchungen wurde das Modell der Thioacetamid- induzierten (TAA) Leberzirrhose der Ratte gewählt. Ähnlich wie beim Menschen, kommt es in diesem Modell durch Entwicklung einer portalen Hypertonie und einer systemischen Hypotonie zur Verschlechterung der Nierenfunktion. Männlichen Wistar-Ratten (N=53) wurde über achtzehn Wochen TAA verabreicht. Ab der achten Woche wurden die Tiere mit Furosemid, SLV329 oder mit beiden Substanzen behandelt. Eine Gruppe wurde nicht behandelt, um den Verlauf der unbehandelten Leberzirrhose zu beobachten. Als Kontrolle dienten Gruppen mit gesunden Ratten und entsprechender Behandlung (N=32). Ergebnisse – Die TAA- Behandlung führte zu einer hohen Mortalität (36%), welche durch die Furosemidbehandlung gesteigert (53%) und durch die SLV329-Behandlung vermindert (16%, p = 0,046 vs. Furosemid-behandelte TAA-Ratten) wurde. TAA führte zur Entwicklung einer ausgeprägten Leberzirrhose mit signifikanter Minderung der Diurese und Verschlechterung der Nierenfunktion (GFR). Sowohl die Furosemidbehandlung als auch die SLV-Behandlung führte bei den TAA- behandelten Tieren zur Erhöhung der Diurese, jedoch bewirkte die Furosemidbehandlung zugleich eine tendenzielle Verminderung der GFR. Dagegen zeigten die SLV329-behandelten Tiere eine starke Tendenz zur Verbesserung der Nierenleistung. Die kombinierte Therapie mit Furosemid und SLV329 führte zu einem stärkeren diuretischen Effekt als die Monotherapien, war aber, ähnlich wie die Furosemidbehandlung, nachteilig für die GFR. Die unterschiedlichen Behandlungen hatten keinen signifikanten Einfluss auf das Ausmaß der Leberzirrhose. In keiner der Gruppen konnten histologisch sichtbare Veränderungen in den Nieren festgestellt werden. Schlussfolgerung – Die vorliegende Studie konnte zeigen, dass die Behandlung mit dem A1R-Antagonisten SLV329 bei der Leberzirrhose der Ratte zu einer Steigerung der Diurese mit einer tendenziellen Verbesserung der Nierenfunktion führt. Die kombinierte Behandlung mit Furosemid und SLV329 hatte eine noch stärkere diuretische Wirkung, allerdings mit einer gleichzeitigen Verschlechterung der Nierenleistung. Diese Ergebnisse deuten darauf hin, dass die SLV329-Monotherapie eine neue Vorgehensweise für die Aszitesbehandlung von Zirrhosepatienten bieten könnte und auch für Prophylaxe und Therapie des HRS eingesetzt werden könnte.Background and Purpose - Hepatorenal syndrome (HRS) is initially a functional renal failure that develops in patients with severe liver disease. Incidence of HRS in patients with decompensated cirrhosis is 8%. In cirrhotic patients with ascites, diuretic therapy is frequently followed by renal function disturbance, which can result in HRS. Adenosine A1 receptors (A1R) are involved in the pathogenesis of HRS due to several mechanisms. Intrahepatic A1R are responsible for the adenosine-mediated hepatorenal reflex. Activation of renal A1R results in cortical vasoconstriction and stimulates reabsorption of sodium and fluid. Inhibition of renal A1R increases urine output without deterioration in glomerular filtration rate (GFR). In patients with congestive heart failure, urine volume increases and there is no deterioration in GFR when A1R antagonist are given in addition to furosemide. In the present study we analyzed the influence of the treatment with SLV329, an A1R antagonist, on renal function and diuresis in a rat model of cirrhosis. Methods – We tested the effect of SLV329 in thioacetamide (TAA) induced rat liver cirrhosis. This model mimics clinical conditions of liver cirrhosis characterized by portal hypertension and systemic hypotension. Cirrhosis was induced in male Wistar rats by TAA administration for eighteen weeks (N=53). After eight weeks, treatment with SLV329 or furosemide or both began. Development of liver cirrhosis was confirmed by measurements of liver enzymes activity and histological analyses of liver tissue. We estimated kidney function by calculation of GFR. Diuresis was assessed on the basis of water intake. The expression of adenosine receptors was determined by Western blot analysis in liver and kidney. Results – TAA treatment was associated with a high mortality (36%) and this was increased by furosemide (53%), whereas SLV329 reduced the mortality (16%, p = 0.046 vs. furosemide treated rats). TAA caused distinctive liver cirrhosis with significant deterioration of diuresis and impairment of renal function (GFR). Furosemide and SLV329 treatment enhanced diuresis significantly in TAA treated rats. However, furosemide led to further reduction of GFR. In contrast SLV329 treatment showed a strong trend towards improvement of renal function. The dual therapy with furosemide and SLV329 produced a stronger diuretic effect than monotherapies with furosemide or SLV329 but affected renal function adversely. Neither furosemide, nor SLV329 influenced the development of liver cirrhosis. No histologically detectable changes in renal tissue could be demonstrated in any treated group. Conclusion - The present study demonstrates that treatment with SLV329, an A1R antagonist, produces a diuretic effect and tends to improve renal function in rats with liver cirrhosis. Combined furosemide and SLV329 treatment additionally increase diuresis but with a decline in GFR. SLV329 monotherapy thus may offer a novel approach to the management of cirrhotic ascites, and could be used for prophylaxis and therapy of HRS as well

Global Overexpression of ET-1 Decreases Blood Pressure - A Systematic Review and Meta-Analysis of ET-1 Transgenic Mice

Background/Aims: ET-1 has independent effects on blood pressure regulation in vivo, it is involved in tubular water and salt excretion, promotes constriction of smooth muscle cells, modulates sympathetic nerve activity, and activates the liberation of nitric oxide. To determine the net effect of these partially counteracting mechanisms on blood pressure, a systematic meta-analysis was performed. Methods: Based on the principles of Cochrane systematic reviews, we searched in major literature databases - MEDLINE (PubMed), Embase, Google Scholar, and the China Biological Medicine Database (CBM-disc) - for articles relevant to the topic of the blood pressure phenotype of endothelin-1 transgenic (ET-1+/+) mice from January 1, 1988 to March 31, 2016. Review Manager Version 5.0 (Rev-Man 5.0) software was applied for statistical analysis. In total thirteen studies reported blood pressure data. Results: The meta-analysis of blood pressure data showed that homozygous ET-1 transgenic mice (ET-1+/+ mice) had a significantly lower blood pressure as compared to WT mice (mean difference: -2.57 mmHg, 95% CI: -4.98∼ -0.16, P = 0.04), with minimal heterogeneity (P = 0.86). A subgroup analysis of mice older than 6 months revealed that the blood pressure difference between ET-1+/+ mice and WT mice was even more pronounced (mean difference: -6.19 mmHg, 95% CI: -10.76∼ -1.62, P = 0.008), with minimal heterogeneity (P = 0.91). Conclusion: This meta-analysis provides robust evidence that global ET-1 overexpression in mice lowers blood pressure in an age-dependent manner. Older ET-1+/+ mice have a somewhat more pronounced reduction of blood pressure

Vascular endothelium derived endothelin-1 is required for normal heart function after chronic pressure overload in mice.

BACKGROUND: Endothelin-1 participates in the pathophysiology of heart failure. The reasons for the lack of beneficial effect of endothelin antagonists in heart failure patients remain however speculative. The anti-apoptotic properties of ET-1 on cardiomyocytes could be a reasonable explanation. We therefore hypothesized that blocking the pro-apoptotic TNF-α pathway using pentoxifylline could prevent the deleterious effect of the lack of ET-1 in a model for heart failure. METHODS: We performed transaortic constriction (TAC) in vascular endothelial cells specific ET-1 deficient (VEETKO) and wild type (WT) mice (n = 5-9) and treated them with pentoxifylline for twelve weeks. RESULTS: TAC induced a cardiac hypertrophy in VEETKO and WT mice but a reduction of fractional shortening could be detected by echocardiography in VEETKO mice only. Cardiomyocyte diameter was significantly increased by TAC in VEETKO mice only. Pentoxifylline treatment prevented cardiac hypertrophy and reduction of fractional shortening in VEETKO mice but decreased fractional shortening in WT mice. Collagen deposition and number of apoptotic cells remained stable between the groups as did TNF-α, caspase-3 and caspase-8 messenger RNA expression levels. TAC surgery enhanced ANP, BNP and bcl2 expression. Pentoxifylline treatment reduced expression levels of BNP, bcl2 and bax. CONCLUSIONS: Lack of endothelial ET-1 worsened the impact of TAC-induced pressure overload on cardiac function, indicating the crucial role of ET-1 for normal cardiac function under stress. Moreover, we put in light a TNF-α-independent beneficial effect of pentoxifylline in the VEETKO mice suggesting a therapeutic potential for pentoxifylline in a subpopulation of heart failure patients at higher risk

ET-1 from endothelial cells is required for complete angiotensin II-induced cardiac fibrosis and hypertrophy

AbstractAimsHypertensive patients develop cardiac hypertrophy and fibrosis with increased stiffness, contractile deficit and altered perfusion. Angiotensin II (AngII) is an important factor in the promotion of this pathology. The effects of AngII are partly mediated by endothelin-1 (ET-1) and transforming growth factor-β. The exact feature of these pathways and the intercellular communications involved remain unclear. In this study, we explored the role of endothelial cell-derived ET-1 in the development of AngII-induced cardiac fibrosis and hypertrophy.Main methodsWe used mice with vascular endothelial cell specific ET-1 deficiency (VEETKO) and their wild type littermates (WT). Mice were infused for one week with AngII (3.2mg/kg/day, n=12) or vehicle (0.15mol/L NaCl and 1mmol/L acetic acid, n=5), using subcutaneous mini-pumps. Hearts were stained with hematoxylin–eosin and masson's trichrome for histology. Cardiac gene expression and protein abundance were measured by Northern Blot, real time PCR and Western Blot.Key findingsAngII-induced cardiac hypertrophy, interstitial and perivascular fibrosis were less pronounced in VEETKO mice compared to WT. Blood pressure increased similarly in both genotypes. Expression of connective tissue growth factor, tumor growth factor-β, collagen I and III in response to AngII required endothelial ET-1. Endothelial ET-1 was also necessary to the elevation in protein kinase C δ abundance and ERK1/2 activation. AngII-induced elevation in PKCε abundance was however ET-1 independent.SignificanceThis study underscores the significance of ET-1 from the vasculature in the process of AngII-induced cardiac hypertrophy and fibrosis, independently from blood pressure. Endothelial ET-1 represents therefore a possible pharmacological target