Runcaciguat, a novel soluble guanylate cyclase activator, shows renoprotection in hypertensive, diabetic, and metabolic preclinical models of chronic kidney disease.

Chronic kidney diseaQueryse (CKD) is associated with oxidative stress which can interrupt the nitric oxide (NO)/soluble guanylyl cyclase (sGC) signaling and decrease cyclic guanosine monophosphate (cGMP) production. Low cGMP concentrations can cause kidney damage and progression of CKD. The novel sGC activator runcaciguat targets the oxidized and heme-free form of sGC, restoring cGMP production under oxidative stress. The purpose of this study is to investigate if runcaciguat could provide an effective treatment for CKD. Runcaciguat was used for the treatment not only in rat CKD models with different etiologies and comorbidities, namely of hypertensive rats, the renin transgenic (RenTG) rat, and angiotensin-supplemented (ANG-SD) rat, but also in rats with diabetic and metabolic CKD, the Zucker diabetic fatty (ZDF) rat. The treatment duration was 2 to 42 weeks and runcaciguat was applied orally in doses from 1 to 10 mg/kg/bid. In these different rat CKD models, runcaciguat significantly reduced proteinuria (urinary protein to creatinine ratio; uPCR). These effects were also significant at doses which did not or only moderately decrease systemic blood pressure. Moreover, runcaciguat significantly decreased kidney injury biomarkers and attenuated morphological kidney damages. In RenTG rats, runcaciguat improved survival rates and markers of heart injury. These data demonstrate that the sGC activator runcaciguat exhibits cardio-renal protection at doses which did not reduce blood pressure and was effective in hypertensive as well as diabetic and metabolic CKD models. These data, therefore, suggest that runcaciguat, with its specific mode of action, represents an efficient treatment approach for CKD and associated CV diseases.

Vascular Protection and Decongestion Without Renin-Angiotensin-Aldosterone System Stimulation Mediated by a Novel Dual-Acting Vasopressin V1a/V2 Receptor Antagonist.

Increased plasma vasopressin levels have been shown to be associated with the progression of congestive heart failure. Vasopressin mediates water retention by renal tubular V2 receptor activation as well as vasoconstriction, cardiac hypertrophy, and fibrosis through V1a receptor activation. Therefore, we developed a novel, dual-acting vasopressin receptor antagonist, BAY 1753011, with almost identical Ki-values of 0.5 nM at the human V1a receptor and 0.6 nM at the human V2 receptor as determined in radioactive binding assays. Renal V2 antagonism by BAY 1753011 was compared with the loop diuretic furosemide in acute diuresis experiments in conscious rats. Similar diuretic efficacy was found with 300-mg/kg furosemide (maximal diuretic response) and 0.1-mg/kg BAY 1753011. Furosemide dose-dependently induced plasma renin and angiotensin I levels, while an equiefficient diuretic BAY 1753011 dose did not activate the renin-angiotensin system. BAY 1753011 dose-dependently decreased the vasopressin-induced expression of the profibrotic/hypertrophic marker plasminogen activator inhibitor-1 and osteopontin in rat cardiomyocytes, while the selective V2 antagonist satavaptan was without any effect. The combined vascular V1a-mediated and renal V2-mediated properties as well as the antihypertrophic/antifibrotic activity enable BAY 1753011 to become a viable treatment option for oral chronic treatment of congestive heart failure.

Urinary cGMP predicts major adverse renal events in patients with mild renal impairment and/or diabetes mellitus before exposure to contrast medium.

BACKGROUND: The use of iodine-based contrast agents entails the risk of contrast induced nephropathy (CIN). Radiocontrast agents elicit the third most common cause of nephropathy among hospitalized patients, accounting for 11-12% of cases. CIN is connected with clinically significant consequences, including increased morbidity, prolonged hospitalization, increased risk of complications, potential need for dialysis, and increased mortality rate. The number of in-hospital examinations using iodine-based contrast media has been significantly increasing over the last decade. In order to protect patients from possible complications of such examinations, new biomarkers are needed that are able to predict a risk of contrast-induced nephropathy. Urinary and plasma cyclic guanosine monophosphate (cGMP) concentrations are influenced by renal function. Urinary cGMP is primarily of renal cellular origin. Therefore, we assessed if urinary cGMP concentration may predict major adverse renal events (MARE) after contrast media exposure during coronary angiography. METHODS: Urine samples were prospectively collected from non-randomized consecutive patients with either diabetes or preexisting impaired kidney function receiving intra-arterial contrast medium (CM) for emergent or elective coronary angiography at the Charité Campus Mitte, University Hospital Berlin. Urinary cGMP concentration in spot urine was analyzed 24 hours after CM exposure. Patients were followed up over 90 days for occurrence of death, initiation of dialysis, doubling of plasma creatinine concentration or MARE. RESULTS: In total, 289 consecutive patients were included into the study. Urine cGMP/creatinine ratio 24 hours before CM exposure expressed as mean±SD was predictive for the need of dialysis (no dialysis: 89.77±92.85 µM/mM, n = 277; need for dialysis: 140.3±82.90 µM/mM, n = 12, p = 0.008), death (no death during follow-up: 90.60±92.50 µM/mM, n = 280; death during follow-up: 169.88±81.52 µM/mM, n = 9; p = 0.002), and the composite endpoint MARE (no MARE: 86.02±93.17 µM/mM, n = 271; MARE: 146.64±74.68 µM/mM, n = 18, p<0.001) during the follow-up of 90 days after contrast media application. cGMP/creatinine ratio stayed significantly increased at values exceeding 120 µM/mM in patients who developed MARE, required dialysis or died. CONCLUSIONS: Urinary cGMP/creatinine ratio ≥ 120 µM/mM before CM exposure is a promising biomarker for the need of dialysis and all-cause mortality 90 days after CM exposure in patients with preexisting renal impairment or diabetes.

Nitric oxide-sensitive guanylyl cyclase stimulation improves experimental heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) can arise from cardiac and vascular remodeling processes following long-lasting hypertension. Efficacy of common HF therapeutics is unsatisfactory in HFpEF. Evidence suggests that stimulators of the nitric oxide-sensitive soluble guanylyl cyclase (NOsGC) could be of use here. We aimed to characterize the complex cardiovascular effects of NOsGC stimulation using NO-independent stimulator BAY 41-8543 in a double-transgenic rat (dTGR) model of HFpEF. We show a drastically improved survival rate of treated dTGR. We observed less cardiac fibrosis, macrophage infiltration, and gap junction remodeling in treated dTGR. Microarray analysis revealed that treatment of dTGR corrected the dysregulateion of cardiac genes associated with fibrosis, inflammation, apoptosis, oxidative stress, and ion channel function toward an expression profile similar to healthy controls. Treatment reduced systemic blood pressure levels and improved endothelium-dependent vasorelaxation of resistance vessels. Further comprehensive in vivo phenotyping showed an improved diastolic cardiac function, improved hemodynamics, and less susceptibility to ventricular arrhythmias. Short-term BAY 41-8543 application in isolated untreated transgenic hearts with structural remodeling significantly reduced the occurrence of ventricular arrhythmias, suggesting a direct nongenomic role of NOsGC stimulation on excitation. Thus, NOsGC stimulation was highly effective in improving several HFpEF facets in this animal model, underscoring its potential value for patients.

Pre-Interventional Kynurenine Predicts Medium-Term Outcome after Contrast Media Exposure Due to Coronary Angiography.

BACKGROUND/AIMS: Contrast induced acute kidney injury (CI-AKI) remains a serious complication of contrast media enhanced procedures like coronary angiography. There is still a lack of established biomarkers that help to identify patients at high risk for short and long-term complications. The aim of the current study was to evaluate plasma kynurenine as a predictive biomarker for CI-AKI and long-term complications, measured by the combined endpoint "major adverse kidney events" (MAKE) up to 120 days after CM application. METHODS: In this prospective cohort study 245 patients undergoing coronary angiography were analyzed. Blood samples were obtained at baseline, 24h and 48h after contrast media (CM) application to diagnose CI-AKI. Patients were followed for 120 days for adverse clinical events including death, the need for dialysis, and a doubling of plasma creatinine. Occurrence of any of these events was summarized in the combined endpoint MAKE. RESULTS: Preinterventional plasma kynurenine was not associated with CI-AKI. Patients who later developed MAKE displayed significantly increased preinterventional plasma kynurenine levels (p<0.0001). ROC analysis revealed that preinterventional kynurenine is highly predictive for MAKE (AUC=0.838; p<0.0001). The optimal cutoff was found at ≥3.5 µmol/L Using this cutoff, the Kaplan-Meier estimator demonstrated that concentrations of plasma kynurenine ≥3.5 µmol/L were significantly associated with a higher prevalence of MAKE until follow up (p<0.0001). This association remained significant in multivariate Cox regression models adjusted for relevant factors of long-term renal outcome. CONCLUSION: Preinterventional plasma kynurenine might serve as a highly predictive biomarker for MAKE up to 120 days after coronary angiography.

Discovery of the Soluble Guanylate Cyclase Stimulator Vericiguat (BAY 1021189) for the Treatment of Chronic Heart Failure.

The first-in-class soluble guanylate cyclase (sGC) stimulator riociguat was recently introduced as a novel treatment option for pulmonary hypertension. Despite its outstanding pharmacological profile, application of riociguat in other cardiovascular indications is limited by its short half-life, necessitating a three times daily dosing regimen. In our efforts to further optimize the compound class, we have uncovered interesting structure-activity relationships and were able to decrease oxidative metabolism significantly. These studies resulting in the discovery of once daily sGC stimulator vericiguat (compound 24, BAY 1021189), currently in phase 3 trials for chronic heart failure, are now reported.

ADMA predicts major adverse renal events in patients with mild renal impairment and/or diabetes mellitus undergoing coronary angiography.

Asymmetric dimethylarginine (ADMA) is a competitive inhibitor of the nitric oxide (NO)-synthase and a biomarker of endothelial dysfunction (ED). ED plays an important role in the pathogenesis of contrast-induced nephropathy (CIN). The aim of our study was to evaluate serum ADMA concentration as a biomarker of an acute renal damage during the follow-up of 90 days after contrast medium (CM) application.Blood samples were obtained from 330 consecutive patients with diabetes mellitus or mild renal impairment immediately before, 24 and 48 hours after the CM application for coronary angiography. The patients were followed for 90 days. The composite endpoints were major adverse renal events (MARE) defined as occurrence of death, initiation of dialysis, or a doubling of serum creatinine concentration.Overall, ADMA concentration in plasma increased after CM application, although, there was no differences between ADMA levels in patients with and without CIN. ADMA concentration 24 hours after the CM application was predictive for dialysis with a specificity of 0.889 and sensitivity of 0.653 at values higher than 0.71 µmol/L (area under the curve: 0.854, 95% confidential interval: 0.767-0.941, P < 0.001). This association remained significant in multivariate Cox regression models adjusted for relevant factors of long-term renal outcome. 24 hours after the CM application, ADMA concentration in plasma was predictive for MARE with a specificity of 0.833 and sensitivity of 0.636 at a value of more than 0.70 µmol/L (area under the curve: 0.750, 95% confidence interval: 0.602-0.897, P = 0.004). Multivariate logistic regression analysis confirmed that ADMA and anemia were significant predictors of MARE. Further analysis revealed that increased ADMA concentration in plasma was highly significant predictor of MARE in patients with CIN. Moreover, patients with CIN and MARE had the highest plasma ADMA levels 24 hours after CM exposure in our study cohort. The impact of ADMA on MARE was independent of such known CIN risk factors as anemia, pre-existing renal failure, pre-existing heart failure, and diabetes.ADMA concentration in plasma is a promising novel biomarker of major contrast-induced nephropathy-associated events 90 days after contrast media exposure.

Soluble guanylate cyclase stimulator riociguat and phosphodiesterase 5 inhibitor sildenafil ameliorate pulmonary hypertension due to left heart disease in mice.

BACKGROUND: Presence of pulmonary hypertension (PH) and right ventricular dysfunction worsens prognosis in patients with chronic heart failure (CHF). Preclinical and clinical studies suggest a role for the impaired nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) signaling pathway in both PH and CHF. Hence, we examined the effects of the NO-sGC-cGMP pathway modulation by the PDE5 inhibitor sildenafil or sGC stimulator riociguat on pulmonary hemodynamics and heart function in a murine model of secondary PH induced by transverse aortic constriction. METHODS: C57Bl/6N mice were subjected to transverse aortic constriction (TAC) for 6weeks to induce left heart failure and secondary PH and were subsequently treated with either sildenafil (100mg/kg/day) or riociguat (10mg/kg/day) or placebo for 2weeks. RESULTS: Six weeks after surgery, TAC induced significant left ventricular hypertrophy and dysfunction associated with development of PH. Treatment with riociguat and sildenafil neither reduced left ventricular hypertrophy nor improved its function. However, both sildenafil and riociguat ameliorated PH, reduced pulmonary vascular remodeling and improved right ventricular function. CONCLUSIONS: Thus, modulation of the NO-sGC-cGMP pathway by the PDE5 inhibitor sildenafil or sGC stimulator riociguat exerts direct beneficial effects on pulmonary hemodynamics and right ventricular function in the experimental model of secondary PH due to left heart disease and these drugs may offer a new therapeutic option for therapy of this condition.

Urinary Vitamin D Binding Protein and KIM-1 Are Potent New Biomarkers of Major Adverse Renal Events in Patients Undergoing Coronary Angiography.

BACKGROUND: Vitamin-D-binding protein (VDBP) is a low molecular weight protein that is filtered through the glomerulus as a 25-(OH) vitamin D 3/VDBP complex. In the normal kidney VDBP is reabsorbed and catabolized by proximal tubule epithelial cells reducing the urinary excretion to trace amounts. Acute tubular injury is expected to result in urinary VDBP loss. The purpose of our study was to explore the potential role of urinary VDBP as a biomarker of an acute renal damage. METHOD: We included 314 patients with diabetes mellitus or mild renal impairment undergoing coronary angiography and collected blood and urine before and 24 hours after the CM application. Patients were followed for 90 days for the composite endpoint major adverse renal events (MARE: need for dialysis, doubling of serum creatinine after 90 days, unplanned emergency rehospitalization or death). RESULTS: Increased urine VDBP concentration 24 hours after contrast media exposure was predictive for dialysis need (no dialysis: 113.06 ± 299.61 ng/ml, n = 303; need for dialysis: 613.07 ± 700.45 ng/ml, n = 11, Mean ± SD, p<0.001), death (no death during follow-up: 121.41 ± 324.45 ng/ml, n = 306; death during follow-up: 522.01 ± 521.86 ng/ml, n = 8; Mean ± SD, p<0.003) and MARE (no MARE: 112.08 ± 302.00 ng/ml, n = 298; MARE: 506.16 ± 624.61 ng/ml, n = 16, Mean ± SD, p<0.001) during the follow-up of 90 days after contrast media exposure. Correction of urine VDBP concentrations for creatinine excretion confirmed its predictive value and was consistent with increased levels of urinary Kidney Injury Molecule-1 (KIM-1) and baseline plasma creatinine in patients with above mentioned complications. The impact of urinary VDBP and KIM-1 on MARE was independent of known CIN risk factors such as anemia, preexisting renal failure, preexisting heart failure, and diabetes. CONCLUSIONS: Urinary VDBP is a promising novel biomarker of major contrast induced nephropathy-associated events 90 days after contrast media exposure.

Chronic Activation of Heme Free Guanylate Cyclase Leads to Renal Protection in Dahl Salt-Sensitive Rats.

The nitric oxide (NO)/soluble guanylate cyclase (sGC)/cyclic guanosine monophasphate (cGMP)-signalling pathway is impaired under oxidative stress conditions due to oxidation and subsequent loss of the prosthetic sGC heme group as observed in particular in chronic renal failure. Thus, the pool of heme free sGC is increased under pathological conditions. sGC activators such as cinaciguat selectively activate the heme free form of sGC and target the disease associated enzyme. In this study, a therapeutic effect of long-term activation of heme free sGC by the sGC activator cinaciguat was investigated in an experimental model of salt-sensitive hypertension, a condition that is associated with increased oxidative stress, heme loss from sGC and development of chronic renal failure. For that purpose Dahl/ss rats, which develop severe hypertension upon high salt intake, were fed a high salt diet (8% NaCl) containing either placebo or cinaciguat for 21 weeks. Cinaciguat markedly improved survival and ameliorated the salt-induced increase in blood pressure upon treatment with cinaciguat compared to placebo. Renal function was significantly improved in the cinaciguat group compared to the placebo group as indicated by a significantly improved glomerular filtration rate and reduced urinary protein excretion. This was due to anti-fibrotic and anti-inflammatory effects of the cinaciguat treatment. Taken together, this is the first study showing that long-term activation of heme free sGC leads to renal protection in an experimental model of hypertension and chronic kidney disease. These results underline the promising potential of cinaciguat to treat renal diseases by targeting the disease associated heme free form of sGC.

Endothelin-1 Overexpression Improves Renal Function in eNOS Knockout Mice.

BACKGROUND/AIMS: To investigate the renal phenotype under conditions of an activated renal ET-1 system in the status of nitric oxide deficiency, we compared kidney function and morphology in wild-type, ET-1 transgenic (ET+/+), endothelial nitric oxide synthase knockout (eNOS-/-) and ET+/+eNOS-/- mice. METHODS: We assessed blood pressure, parameters of renal morphology, plasma cystatin C, urinary protein excretion, expression of genes associated with glomerular filtration barrier and tissue remodeling, and plasma metabolites using metabolomics. RESULTS: eNOS-/- and ET+/+eNOS-/- mice developed hypertension. Osteopontin, albumin and protein excretion were increased in eNOS-/- and restored in ET+/+eNOS-/- animals. All genetically modified mice developed renal interstitial fibrosis and glomerulosclerosis. Genes involved in tissue remodeling (serpine1, TIMP1, Col1a1, CCL2) were up-regulated in eNOS-/-, but not in ET+/+eNOS-/- mice. Plasma levels of free carnitine and acylcarnitines, amino acids, diacyl phosphatidylcholines, lysophosphatidylcholines and hexoses were descreased in eNOS-/- and were in the normal range in ET+/+eNOS-/- mice. CONCLUSION: eNOS-/- mice developed renal dysfunction, which was partially rescued by ET-1 overexpression in eNOS-/- mice. The metabolomics results suggest that ET-1 overexpression on top of eNOS knockout is associated with a functional recovery of mitochondria (rescue effect in ß-oxidation of fatty acids) and an increase in antioxidative properties (normalization of monounsaturated fatty acids levels).

Finerenone, a novel selective nonsteroidal mineralocorticoid receptor antagonist protects from rat cardiorenal injury.

Pharmacological blockade of the mineralocorticoid receptor (MR) ameliorates end-organ damage in chronic heart failure. However, the clinical use of available steroidal MR antagonists is restricted because of concomitant hyperkalemia especially in patients with diminished kidney function. We have recently identified a novel nonsteroidal MR antagonist, finerenone, which uniquely combines potency and selectivity toward MR. Here, we investigated the tissue distribution and chronic cardiorenal end-organ protection of finerenone in comparison to the steroidal MR antagonist, eplerenone, in 2 different preclinical rat disease models. Quantitative whole-body autoradiography revealed that [C]-labeled finerenone equally distributes into rat cardiac and renal tissues. Finerenone treatment prevented deoxycorticosterone acetate-/salt-challenged rats from functional as well as structural heart and kidney damage at dosages not reducing systemic blood pressure. Finerenone reduced cardiac hypertrophy, plasma prohormone of brain natriuretic peptide, and proteinuria more efficiently than eplerenone when comparing equinatriuretic doses. In rats that developed chronic heart failure after coronary artery ligation, finerenone (1 mg·kg·d), but not eplerenone (100 mg·kg·d) improved systolic and diastolic left ventricular function and reduced plasma prohormone of brain natriuretic peptide levels. We conclude that finerenone may offer end-organ protection with a reduced risk of electrolyte disturbances.

Urinary ET-1 excretion after exposure to radio-contrast media in diabetic patients and patients with preexisting mild impaired renal function.

AIMS: Contrast media-induced nephropathy (CIN) is associated with increased morbidity and mortality. The renal endothelin system has been associated with disease progression of various acute and chronic renal diseases. However, robust data coming from adequately powered prospective clinical studies analyzing the short and long-term impacts of the renal ET system in patients with CIN are missing so far. We thus performed a prospective study addressing this topic. MAIN METHODS: We included 327 patients with diabetes or renal impairment undergoing coronary angiography. Blood and spot urine were collected before and 24 h after contrast media (CM) application. Patients were followed for 90 days for major clinical events like need for dialysis, unplanned rehospitalization or death. KEY FINDINGS: The concentration of ET-1 and the urinary ET-1/creatinine ratio decreased in spot urine after CM application (ET-1 concentration: 0.91±1.23 pg/ml versus 0.63±1.03 pg/ml, p<0.001; ET-1/creatinine ratio: 0.14±0.23 versus 0.09±0.19, p<0.001). The urinary ET-1 concentrations in patients with CIN decreased significantly more than in patients without CIN (-0.26±1.42 pg/ml vs. -0.79±1.69 pg/ml, p=0.041), whereas the decrease of the urinary ET-1/creatinine ratio was not significantly different (non-CIN patients: -0.05±0.30; CIN patients: -0.11±0.21, p=0.223). Urinary ET-1 concentrations as well as the urinary ET-1/creatinine ratio were not associated with clinical events (need for dialysis, rehospitalization or death) during the 90 day follow-up after contrast media exposure. However, the urinary ET-1 concentration and the urinary ET-1/creatinine ratio after CM application were higher in those patients who had a decrease of GFR of at least 25% after 90 days of follow-up. SIGNIFICANCE: In general the ET-1 system in the kidney seems to be down-regulated after contrast media application in patients with moderate CIN risk. Major long-term complications of CIN (need for dialysis, rehospitalization or death) are not associated with the renal ET system.

The enzymatic activity of the VEGFR2 receptor for the biosynthesis of dinucleoside polyphosphates.

The group of dinucleoside polyphosphates encompasses a large number of molecules consisting of two nucleosides which are connected by a phosphate chain of variable length. While the receptors activated by dinucleoside polyphosphates as well as their degradation have been studied in detail, its biosynthesis has not been elucidated so far. Since endothelial cells released the dinucleoside polyphosphate uridine adenosine tetraphosphate (Up4A), we tested cytosolic proteins of human endothelial cells obtained from dermal vessels elicited for enzymatic activity. When incubated with ADP and UDP, these cells showed increasing concentrations of Up4A. The underlying enzyme was isolated by chromatography and the mass spectrometric analysis revealed that the enzymatic activity was caused by the vascular endothelial growth factor receptor 2 (VEGFR2). Since VEGFR2 but neither VEGFR1 nor VEGFR3 were capable to synthesise dinucleoside polyphosphates, Tyr-1175 of VEGFR2 is most likely essential for the enzymatic activity of interest. Further, VEGFR2-containing cells like HepG2, THP-1 and RAW264.7 were capable of synthesising dinucleoside polyphosphates. VEGFR2-transfected HEK 293T/17 but not native HEK 293T/17 cells synthesised dinucleoside polyphosphates in vivo too. The simultaneous biosynthesis of dinucleoside polyphosphates could amplify the response to VEGF, since dinucleoside polyphosphates induce cellular growth via P2Y purinergic receptors. Thus the biosynthesis of dinucleoside polyphosphates by VEGFR2 may enhance the proliferative response to VEGF. Given that VEGFR2 is primarily expressed in endothelial cells, the biosynthesis of dinucleoside polyphosphates is mainly located in the vascular system. Since the vasculature is also the main site of action of dinucleoside polyphosphates, activating vascular purinoceptors, blood vessels appear as an autocrine system with respect to dinucleoside polyphosphates. We conclude that VEGFR2 receptor is capable of synthesising dinucleoside polyphosphates. These mediators may modulate the effects of VEGFR2 due to their proliferative effects.

Riociguat reduces infarct size and post-infarct heart failure in mouse hearts: insights from MRI/PET imaging.

AIM: Stimulation of the nitric oxide (NO)--soluble guanylate (sGC)--protein kinase G (PKG) pathway confers protection against acute ischaemia/reperfusion injury, but more chronic effects in reducing post-myocardial infarction (MI) heart failure are less defined. The aim of this study was to not only determine whether the sGC stimulator riociguat reduces infarct size but also whether it protects against the development of post-MI heart failure. METHODS AND RESULTS: Mice were subjected to 30 min ischaemia via ligation of the left main coronary artery to induce MI and either placebo or riociguat (1.2 µmol/l) were given as a bolus 5 min before and 5 min after onset of reperfusion. After 24 hours, both, late gadolinium-enhanced magnetic resonance imaging (LGE-MRI) and (18)F-FDG-positron emission tomography (PET) were performed to determine infarct size. In the riociguat-treated mice, the resulting infarct size was smaller (8.5 ± 2.5% of total LV mass vs. 21.8% ± 1.7%. in controls, p = 0.005) and LV systolic function analysed by MRI was better preserved (60.1% ± 3.4% of preischaemic vs. 44.2% ± 3.1% in controls, p = 0.005). After 28 days, LV systolic function by echocardiography treated group was still better preserved (63.5% ± 3.2% vs. 48.2% ± 2.2% in control, p = 0.004). CONCLUSION: Taken together, mice treated acutely at the onset of reperfusion with the sGC stimulator riociguat have smaller infarct size and better long-term preservation of LV systolic function. These findings suggest that sGC stimulation during reperfusion therapy may be a powerful therapeutic treatment strategy for preventing post-MI heart failure.

Early urinary and plasma biomarkers for experimental diabetic nephropathy.

BACKGROUND: As the prevalence of diabetes rises, its complications such as diabetic nephropathy affect an increaseing number of patients. Consequently, the need for biomarkers in rodent models which reflect the stage and course of diabetic nephropathy is high. This article focuses on Heart-type fatty acid binding protein (H-FABP), osteopontin (OPN), nephrin, and Neutrophil gelatinase-associated lipocalin (NGAL) in urine, and kidney injury molecule (KIM)-1, clusterin, and tissue inhibitior of metalloproteinases (TIMP) 1 in plasma in uni-nephrectomized rats with streptocotozin-induced type 1 diabetes mellitus, a common animal model to explore renal impairment in the setting of diabetes mellitus. METHODS: 23 male Wistar rats were uni-nephrectomized and subsequently divided into two study groups. The diabetic group received streptozotocin (STZ) via tail-vein injection, the non-diabetic group received citrate buffer without STZ. Subsequently, blood glucose, body weight, and blood pressure were checked regularly. After 18 weeks, animals were placed in metabolic cages, blood and urine obtained and subsequently organs were harvested after sacrifice. RESULTS: Blood glucose levels were highly increased in diabetic animals throughout the experiment, whereas systolic blood pressure did not differ between the study groups. At study end, classical biomarkers such as urinary albumin and protein and plasma cystatin c were only slightly but not significantly different between groups indicating a very early disease state. In contrast, urinary excretion of H-FABP, OPN, nephrin, and NGAL were highly increased in diabetic animals with a highly significant p-value (p < 0.01 each) compared to non-diabetic animals. In plasma, differences were found for calbindin, KIM-1, clusterin, TIMP-1, and OPN. These findings were confirmed by means of the area under the receiver operating characteristic curve (ROC-AUC) analysis. CONCLUSIONS: In summary, our study revealed elevated levels of new plasma and urinary biomarkers (urinary osteopontin, urinary nephrin, urinary NGAL, urinary H-FABP, plasma KIM-1, plasma TIMP-1) in uni-nephrectomized diabetic rats, an established rat model of diabetic nephropathy. These biomarkers appeared even before the classical biomarkers of diabetic nephropathy such as albuminuria and urinary protein excretion. The new biomarkers might offer an advantage to urinary albumin and plasma cystatin c with respect to early detection.

Effects of stimulation of soluble guanylate cyclase on diabetic nephropathy in diabetic eNOS knockout mice on top of angiotensin II receptor blockade.

The prevalence of diabetes mellitus and its complications, such as diabetic nephropathy (DN), is rising worldwide and prevention and treatment are therefore becoming increasingly important. Therapy of DN is particularly important for patients who do not adequately respond to angiotensin receptor blocker (ARB) treatment. Novel approaches include the stimulation of soluble guanylate cyclase (sGC) as it is reported to have beneficial effects on cardiac and renal damage. We aimed to investigate the effects of the sGC stimulator riociguat and ARB telmisartan on kidney function and structure in a hypertensive model of diabetic nephropathy. Seventy-six diabetic male eNOS knockout C57BL/6J mice were randomly divided after having received streptozotocin: telmisartan (1 mg/kg/d), riociguat (3 mg/kg/d), riociguat+telmisartan (3+1 mg/kg/d), and vehicle. Fourteen mice were used as non-diabetic controls. Treatment duration was 11 weeks. Glucose concentrations were increased and similar in all diabetic groups. Telmisartan insignificantly reduced blood pressure by 5.9 mmHg compared with diabetic controls (111.2±2.3 mmHg vs. 117.1±2.2 mmHg; p = 0.071). Treatment with riociguat both alone and in combination with telmisartan led to a significant reduction of blood pressure towards diabetic vehicle (105.2±2.5 mmHg and 105.0±3.2 mmHg, respectively, vs. 117.1±2.2 mmHg). Combined treatment also significantly decreased albuminuria compared with diabetic controls (47.3±9.6 µg/24 h vs. 170.8±34.2 µg/24 h; p = 0.002) reaching levels similar to those of non-diabetic controls (34.4±10.6 µg/24 h), whereas the reduction by single treatment with either telmisartan (97.8±26.4 µg/24 h) or riociguat (97.1±15.7 µg/24 h) was not statistically significant. The combination treatment led to a significant (p<0.01) decrease of tissue immunoreactivity of malondialdehyde, as consequence of reduced oxidative stress. In conclusion, stimulation of sGC significantly reduced urinary albumin excretion in diabetic eNOS knockout mice treated already with ARB. Thus, this new drug class on top of standard ARBs administration may offer a new therapeutic approach for patients resistant to ARB treatment.

The soluble guanylate cyclase stimulator riociguat ameliorates pulmonary hypertension induced by hypoxia and SU5416 in rats.

BACKGROUND: The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) signal-transduction pathway is impaired in many cardiovascular diseases, including pulmonary arterial hypertension (PAH). Riociguat (BAY 63-2521) is a stimulator of sGC that works both in synergy with and independently of NO to increase levels of cGMP. The aims of this study were to investigate the role of NO-sGC-cGMP signaling in a model of severe PAH and to evaluate the effects of sGC stimulation by riociguat and PDE5 inhibition by sildenafil on pulmonary hemodynamics and vascular remodeling in severe experimental PAH. METHODS AND RESULTS: Severe angioproliferative PAH was induced in rats by combined exposure to the vascular endothelial growth factor receptor antagonist SU5416 and hypoxia (SUHx). Twenty-one days thereafter rats were randomized to receive either riociguat (10 mg/kg/day), sildenafil (50 mg/kg/day) or vehicle by oral gavage, for 14 days until the day of the terminal hemodynamic measurements. Administration of riociguat or sildenafil significantly decreased right ventricular systolic pressure (RVSP). Riociguat significantly decreased RV hypertrophy (RVH) (0.55 ± 0.02, p<0.05), increased cardiac output (60.8 ± .8 mL/minute, p<0.05) and decreased total pulmonary resistance (4.03 ± 0.3 mmHg min(-1) ml(-1) 100 g BW, p<0.05), compared with sildenafil and vehicle. Both compounds significantly decreased the RV collagen content and improved RV function, but the effects of riociguat on tricuspid annular plane systolic excursion and RV myocardial performance were significantly better than those of sildenafil (p<0.05). The proportion of occluded arteries was significantly lower in animals receiving riociguat than in those receiving vehicle (p<0.05); furthermore, the neointima/media ratio was significantly lower in those receiving riociguat than in those receiving sildenafil or vehicle (p<0.05). CONCLUSION: Riociguat and sildenafil significantly reduced RVSP and RVH, and improved RV function compared with vehicle. Riociguat had a greater effect on hemodynamics and RVH than sildenafil.

Relationship between magnesium and clinical biomarkers on inhibition of vascular calcification.

BACKGROUND: Arteriosclerosis and cardiovascular disease are strongly associated with vascular calcification. Hyperphosphatemia is an essential risk factor for increased vascular calcification. End-stage renal disease (ESRD) patients could serve as an in vivo model for accelerated calcification. This study focuses on the most likely protective effects of magnesium ion (Mg(2+)) on phosphate-induced vascular calcification ex vivo/in vitro. Furthermore, plasma Mg(2+) concentrations of ESRD and healthy controls were investigated for association with surrogate parameters of vascular calcification in vivo. METHODS: Aortic segments of male Wistar-Kyoto rats were incubated and the phosphate concentration of the medium was elevated. The aortic segments were incubated in the absence and presence of MgCl(2); tissue calcification was quantified by different methods. Serum Mg(2+) concentrations of patients with chronic kidney disease (CKD stage 5; ESRD) and patients without CKD (controls) were associated with carotid intima media thickness (IMT) and aortic pulse wave velocity (PWV) as surrogate parameter for arteriosclerosis and arterial stiffening. RESULTS: Incubation of aortic segments in the presence of ß-glycerophosphate and NaH(2)PO(4) caused an increased tissue Ca(2+) deposition compared to control conditions. This increased amount of Ca(2+) in the aortic rings was significantly decreased in the presence of Mg(2+). In CKD patients, but not in controls, magnesium serum concentration was associated with the IMT of the carotid arteries. In addition, CKD patients with higher magnesium serum concentration had a significantly lower PWV. DISCUSSION AND CONCLUSION: Elevated phosphate concentrations in the culture media induce ex vivo/in vitro medial calcification in intact rat aortic rings in the presence of alkaline phosphatase. Mg(2+) ions reduced ex vivo/in vitro vascular calcification despite increased phosphate concentration. This hypothesis is additionally based on the fact that CKD patients with high Mg(2) serum levels had significantly lower IMT and PWV values, which may result in a lower risk for cardiovascular events and mortality in these patients. Therefore, Mg(2+) supplementation may be an option for treatment and prevention of vascular calcification resulting in a reduction of cardiovascular events in CKD patients.

Soluble guanylate cyclase stimulation prevents fibrotic tissue remodeling and improves survival in salt-sensitive Dahl rats.

BACKGROUND: A direct pharmacological stimulation of soluble guanylate cyclase (sGC) is an emerging therapeutic approach to the management of various cardiovascular disorders associated with endothelial dysfunction. Novel sGC stimulators, including riociguat (BAY 63-2521), have a dual mode of action: They sensitize sGC to endogenously produced nitric oxide (NO) and also directly stimulate sGC independently of NO. Little is known about their effects on tissue remodeling and degeneration and survival in experimental malignant hypertension. METHODS AND RESULTS: Mortality, hemodynamics and biomarkers of tissue remodeling and degeneration were assessed in Dahl salt-sensitive rats maintained on a high salt diet and treated with riociguat (3 or 10 mg/kg/d) for 14 weeks. Riociguat markedly attenuated systemic hypertension, improved systolic heart function and increased survival from 33% to 85%. Histological examination of the heart and kidneys revealed that riociguat significantly ameliorated fibrotic tissue remodeling and degeneration. Correspondingly, mRNA expression of the pro-fibrotic biomarkers osteopontin (OPN), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and plasminogen activator inhibitor-1 (PAI-1) in the myocardium and the renal cortex was attenuated by riociguat. In addition, riociguat reduced plasma and urinary levels of OPN, TIMP-1, and PAI-1. CONCLUSIONS: Stimulation of sGC by riociguat markedly improves survival and attenuates systemic hypertension and systolic dysfunction, as well as fibrotic tissue remodeling in the myocardium and the renal cortex in a rodent model of pressure and volume overload. These findings suggest a therapeutic potential of sGC stimulators in diseases associated with impaired cardiovascular and renal functions.