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Elderly patients have increased susceptibility to acute kidney injury (AKI). Long noncoding RNAs (lncRNA) are key regulators of cellular processes, and have been implicated in both aging and AKI. Our aim was to study the effects of aging and ischemia–reperfusion injury (IRI) on the renal expression of lncRNAs. Adult and old (10- and 26–30-month-old) C57BL/6 N mice were subjected to unilateral IRI followed by 7 days of reperfusion. Renal expression of 90 lncRNAs and mRNA expression of injury, regeneration, and fibrosis markers was measured by qPCR in the injured and contralateral control kidneys. Tubular injury, regeneration, and fibrosis were assessed by histology. Urinary lipocalin-2 excretion was increased in old mice prior to IRI, but plasma urea was similar. In the control kidneys of old mice tubular cell necrosis and apoptosis, mRNA expression of kidney injury molecule-1, fibronectin-1, p16, and p21 was elevated. IRI increased plasma urea concentration only in old mice, but injury, regeneration, and fibrosis scores and their mRNA markers were similar in both age groups. AK082072 and Y lncRNAs were upregulated, while H19 and RepA transcript were downregulated in the control kidneys of old mice. IRI upregulated Miat, Igf2as, SNHG5, SNHG6, RNCR3, Malat1, Air, Linc1633, and Neat1 v1, while downregulated Linc1242. LncRNAs H19, AK082072, RepA transcript, and Six3os were influenced by both aging and IRI. Our results indicate that both aging and IRI alter renal lncRNA expression suggesting that lncRNAs have a versatile and complex role in aging and kidney injury. An Ingenuity Pathway Analysis highlighted that the most downregulated H19 may be linked to aging/senescence through p53.

According to our experience the 12-lead electrocardiogram (ECG) may be used to estimate the pretest probability of acute pulmonary embolism (acPE). To this end, we devised a novel ECG score (nECGs) composed of 5 known ECG criteria, best characterizing the key pathogenetic steps of acPE. A retrospective derivation cohort including 136 patients with acPE and a prospective validation cohort including 149 consecutive patients were used to devise and validate the nECGs. The latter cohort consisted of 76 patients with acPE and 73 controls presenting with characteristic symptoms of acPE, in whom the work-up ruled out acPE. We compared the diagnostic value of our nECGs with those of another ECG score (Daniel-ECG-score) and of the best prediction rules (3 Wells score and 2 Geneva score variants). The sensitivity (98.7%), negative predictive value (98%), test accuracy (84.4%) and the negative likelihood ratio (LR) (0.019) of the nECGs were superior to those of all other investigated methods. There was no between-groups difference in the positive LR. The specificity (69%) of the nECGs was inferior to those of the Daniel-ECG-score and Wells scores and did not differ or was superior to those of the Geneva score variants. The positive predictive value (77.3%) of the nECGs was superior to those of the 2 Geneva scores and did not differ from those of the other methods. In conclusion, the nECGs due to its superior sensitivity, negative predictive value, test accuracy, and negative LR estimated the pretest probability of acPE better than the Daniel-ECG-score and the prediction rules.

Purpose Undesirable complement (C) activation by nanomedicines can entail an adverse immune reaction known as C activation-related pseudoallergy (CARPA) in sensitive patients. The syndrome includes cardiopulmonary, hemodynamic, and a variety of other physiological changes that have been well described in man, pigs, dogs, and rats. However, the information on CARPA is scarce and ambiguous in mice, a species widely used in preclinical studies. The present study aimed to fill this gap by exploring signs of CARPA in mice following i.v. administration of AmBisome and Abelcet, which are nano-formulations of Amphotericin B with high risk to cause CARPA. Materials and methods Anesthetized NMRI mice were intravenously injected with liposomal amphotericin B (Abelcet and AmBisome; 30-300 mg phospholipid/kg), drug-free high cholesterol multilamellar vesicles (HC-MLV), and positive controls, cobra venom factor (CVF) and zymosan, followed by the measurement of blood pressure (BP), heart rate, white blood cell, and platelet counts and plasma thromboxane B2 (TXB2) levels. C activation was assessed by C3a ELISA, a C3 consumption assay (PAN-C3) and a modified sheep red blood cell hemolytic assay. Results All test agents, except HC-MLV, caused transient hypertension, thrombocytopenia, and elevation of plasma TXB2, which were paralleled by significant rises of plasma C3a in CVF and zymosan-treated animals, wherein the initial hypertension turned into hypotension and shock. Abelcet and AmBisome caused minor, delayed rise of C3a that was not associated with hypertension. The C3a receptor inhibitor SB-290157 attenuated the hypertension caused by Abelcet and decreased the BP thereafter. Conclusion The parallelism between C3a anaphylatoxin production and severity of physiological changes caused by the different agents is consistent with CARPA underlying these changes. Although the reactive dose of liposomal phospholipids was substantially higher than that in other species (pigs, dogs), the mouse seems suitable for studying the mechanism of hypersensitivity reactions to liposomal formulations of amphotericin B, a frequent side effect of these drugs.

Background: Organ protection for transplantation is perfusion with ice-cold preservation solutions, although saline is also used in animal experiments and living donor transplantations. However, ice-cold perfusion can contribute to initial graft injury. Our aim was to test if cytoskeletal damage of parenchymal cells is caused by saline itself or by the ice-cold solution. Methods: F344 rat kidneys were flushed with cold (4 &deg, C) saline, ischemic and sham kidneys were not perfused. In a separate set, F344 kidneys were flushed with saline or preservation solution at 4 or 15 &deg, C. Ischemia time was 30 min. Results: Renal injury was significantly more severe following cold ischemia (CI) than after ischemia-reperfusion without flushing (ischemia/reperfusion (I/R)). Functional and morphologic damage was accompanied by severe loss of ezrin from glomerular and tubular epithelial cells after CI. Moreover, saline caused serious injury independently from its temperature, while the perfusion solution was more beneficial, especially at 4 &deg, C. Conclusions: Flushing the kidney with ice-cold saline can cause more severe injury than ischemia-reperfusion at body temperature even during a short (30 min) ischemia. Saline perfusion can prolong recovery from ischemia in kidney transplantation, which can be prevented by using preservation solutions.

Intravenous administration of lipid-based nanodrugs can cause hypersensitivity, also known as infusion reactions (IRs), that can be attenuated by slow infusion in adult patients. We studied the role of infusion rate and complement (C) activation in IRs in pediatric patients treated with Abelcet, and also in anesthetized rats. IRs were observed in 6 out of 10 (60%) patients who received Abelcet infusion in 4 h or less, while no patients who received the infusion in 6 h showed C activation or IRs. The rat model indicated an inverse relationship between infusion speed and Abelcet-induced hypotension, taken as an experimental endpoint of IRs, while the rise of C3a in blood, an index of C activation, directly correlated with hypotension. The results suggest that pediatric patients are more prone to produce IRs, and that the optimal infusion time of Abelcet may be much longer than the presently recommended 2 h.

Background Current cardiac resynchronization therapy (CRT) works by pacing the latest activated left ventricular (LV) site. We hypothesized that the greater nonresponse rate of patients with nonspecific intraventricular conduction disturbance (NICD) than with left bundle branch block (LBBB) pattern to CRT might be due, besides less dyssynchrony, to the inability of the current CRT technique, devised to eliminate dyssynchrony caused by LBBB pattern, to eliminate dyssynchrony in some patients with NICD pattern, because their latest activated LV site is far away from that in LBBB. Methods We devised a novel surface ECG method to estimate the approximate location of the latest activated LV site based on the principle that the resultant ST vector of secondary ST segment alterations associated with wide QRS complexes is directed 180o away from the latest activated LV site. By measuring the amplitude and polarity of secondary ST segment alterations in two optional frontal and horizontal plane ECG leads and using a software, we determined the resultant 3D spatial secondary ST vector in 88 patients with LBBB and 57 patients with NICD patterns and heart failure. To validate the ECG method, we also estimated the latest activated LV region by echocardiography using 3D parametric imaging and 2D speckle tracking in 16 LBBB and 13 NICD patients. Patients with NICD pattern were subdivided according to their non-overlapping frontal plane resultant secondary ST vector ranges to NICD-1 (n=35) and NICD-2 (n=22) subgroups. Results The resultant 3D spatial secondary ST vector coordinates in the LBBB group were: (x axis: −0.228 mV, y axis: −0.062 mV, z axis: 0.63 mV); in the NICD-1 and NICD-2 subgroups: (x: 0.154 and 0.198 mV, y: −0.198 and 0.162 mV, z: 0.422 and 0.398 mV respectively). Consequently the latest activated LV sites were located leftward, posterosuperior in the LBBB group, right, posterosuperior in the NICD-1 and right, posteroinferior in the NICD-2 subgroups. The latest activated LV region determined by ECG and echocardiography matched in all patients, except 1. Conclusions The latest activated LV site was at the expected position in the LBBB group, while it was at an almost opposite site in the NICD-2 group [22/57 (39%)]. Thus, one potential reason for the unfavorable response to CRT, occurring in approx. 40% of patients with NICD pattern with a QRS duration of 120–149 ms in randomized studies, is that the current CRT technique using a left posterolateral LV electrode position may not be able to eliminate dyssynchrony in these patients. Funding Acknowledgement Type of funding source: None

(1) Background: Lipopolysaccharide (LPS)-induced systemic inflammation is associated with septic acute kidney injury (AKI). We investigated the time-dependent miRNA expression changes in the kidney caused by LPS. (2) Methods: Male outbred NMRI mice were injected with LPS and sacrificed at 1.5 and 6 h (40 mg/kg i.p., early phase, EP) or at 24 and 48 h (10 mg/kg i.p., late phase, LP). The miRNA profile was established using miRCURY LNA&trade, microarray and confirmed with qPCR. Total renal proteome was analyzed by LC-MS/MS (ProteomeXchange: PXD014664). (3) Results: Septic AKI was confirmed by increases in plasma urea concentration and in renal TNF-&alpha, and IL-6 mRNA expression. Most miRNAs were altered at 6 and 24 h and declined by 48 h. In EP miR-762 was newly identified and validated and was the most elevated miRNA. The predicted target of miR-762, Ras related GTPase 1B (Sar1b) was downregulated. In LP miR-21a-5p was the most influenced miRNA followed by miR-451a, miR-144-3p, and miR-146a-5p. Among the potential protein targets of the most influenced miRNAs, only aquaporin-1, a target of miR-144-3p was downregulated at 24 h. (4) Conclusion: Besides already known miRNAs, septic AKI upregulated miR-762, which may regulate GTP signaling, and miR-144-3p and downregulated its target, aquaporin-1.

(1) Background: Successful treatment of acute kidney injury (AKI)-induced chronic kidney disease (CKD) is unresolved. We aimed to characterize the time-course of changes after contralateral nephrectomy (Nx) in a model of unilateral ischemic AKI-induced CKD with good translational utility. (2) Methods: Severe (30 min) left renal ischemia-reperfusion injury (IRI) or sham operation (S) was performed in male Naval Medical Research Institute (NMRI) mice followed by Nx or S one week later. Expression of proinflammatory, oxidative stress, injury and fibrotic markers was evaluated by RT-qPCR. (3) Results: Upon Nx, the injured kidney hardly functioned for three days, but it gradually regained function until day 14 to 21, as demonstrated by the plasma urea. Functional recovery led to a drastic reduction in inflammatory infiltration by macrophages and by decreases in macrophage chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-&alpha, ) mRNA and most injury markers. However, without Nx, a marked upregulation of proinflammatory (TNF-&alpha, IL-6, MCP-1 and complement-3 (C3)), oxidative stress (nuclear factor erythroid 2-related factor 2, NRF2) and fibrosis (collagen-1a1 (Col1a1) and fibronectin-1 (FN1)) genes perpetuated, and the injured kidney became completely fibrotic. Contralateral Nx delayed the development of renal failure up to 20 weeks. (4) Conclusion: Our results suggest that macrophage activation is involved in postischemic renal fibrosis, and it is drastically suppressed by contralateral nephrectomy ameliorating progression.

Background and purpose Incidence and severity of obesity are increasing worldwide, however, efficient and safe pharmacological treatments are not yet available. Certain MAO inhibitors reduce body weight, although their effects on metabolic parameters have not been investigated. Here, we have assessed effects of a widely used, selective MAO-B inhibitor, selegiline, on metabolic parameters in a rat model of diet-induced obesity. Experimental approach Male Long-Evans rats were given control (CON) or a high-fat (20%), high-sucrose (15%) diet (HFS) for 25 weeks. From week 16, animals were injected s.c. with 0.25 mg·kg-1 selegiline (CON + S and HFS + S) or vehicle (CON, HFS) once daily. Whole body, subcutaneous and visceral fat was measured by CT, and glucose and insulin tolerance were tested. Expression of glucose transporters and chemokines was assessed by quantitative RT-PCR. Key results Selegiline decreased whole body fat, subcutaneous- and visceral adiposity, measured by CT and epididymal fat weight in the HFS group, compared with HFS placebo animals, without influencing body weight. Oral glucose tolerance and insulin tolerance tests showed impaired glucose homeostasis in HFS and HFS + S groups, although insulin levels in plasma and pancreas were unchanged. HFS induced expression of Srebp-1c, Glut1 and Ccl3 in adipose tissue, which were alleviated by selegiline. Conclusions and implications Selegiline reduced adiposity, changes in adipose tissue energy metabolism and adipose inflammation induced by HFS diet without affecting the increased body weight, impairment of glucose homeostasis, or behaviour. These results suggest that selegiline could mitigate harmful effects of visceral adiposity.

(1) Background: Ischemia reperfusion (IR) is the leading cause of acute kidney injury (AKI) and results in predisposition to chronic kidney disease. We demonstrated that delayed contralateral nephrectomy (Nx) greatly improved the function of the IR-injured kidney and decelerated fibrosis progression. Our aim was to identify microRNAs (miRNA/miR) involved in this process. (2) Methods: NMRI mice were subjected to 30 min of renal IR and one week later to Nx/sham surgery. The experiments were conducted for 7–28 days after IR. On day 8, multiplex renal miRNA profiling was performed. Expression of nine miRNAs was determined with qPCR at all time points. Based on the target prediction, plexin-A2 and Cd2AP were measured by Western blot. (3) Results: On day 8 after IR, the expression of 20/1195 miRNAs doubled, and 9/13 selected miRNAs were upregulated at all time points. Nx reduced the expression of several ischemia-induced pro-fibrotic miRNAs (fibromirs), such as miR-142a-duplex, miR-146a-5p, miR-199a-duplex, miR-214-3p and miR-223-3p, in the injured kidneys at various time points. Plexin-A2 was upregulated by IR on day 10, while Cd2AP was unchanged. (4) Conclusion: Nx delayed fibrosis progression and decreased the expression of ischemia-induced fibromirs. The protein expression of plexin-A2 and Cd2AP is mainly regulated by factors other than miRNAs.

Decreased soluble guanylate cyclase activity and cGMP levels in diabetic kidneys were shown to influence the progression of nephropathy. The regulatory effects of soluble guanylate cyclase activators on renal signaling pathways are still unknown, we therefore investigated the renal molecular effects of the soluble guanylate cyclase activator cinaciguat in type-1 diabetic (T1DM) rats. Male adult Sprague-Dawley rats were divided into 2 groups after induction of T1DM with 60 mg/kg streptozotocin: DM, untreated (DM, n = 8) and 2) DM + cinaciguat (10 mg/kg per os daily, DM-Cin, n = 8). Non-diabetic untreated and cinaciguat treated rats served as controls (Co (n = 10) and Co-Cin (n = 10), respectively). Rats were treated for eight weeks, when renal functional and molecular analyses were performed. Cinaciguat attenuated the diabetes induced proteinuria, glomerulosclerosis and renal collagen-IV expression accompanied by 50% reduction of TIMP-1 expression. Cinaciguat treatment restored the glomerular cGMP content and soluble guanylate cyclase expression, and ameliorated the glomerular apoptosis (TUNEL positive cell number) and podocyte injury. These effects were accompanied by significantly reduced TGF-ß overexpression and ERK1/2 phosphorylation in cinaciguat treated diabetic kidneys. We conclude that the soluble guanylate cyclase activator cinaciguat ameliorated diabetes induced glomerular damage, apoptosis, podocyte injury and TIMP-1 overexpression by suppressing TGF-ß and ERK1/2 signaling.

Background/Aims: Diabetic nephropathy remains a major clinical problem. The effects of prorenin might be adverse, but the literature data are controversial. We compared the renal effects of the (pro)renin receptor ((P)RR) blockade and angiotensin converting enzyme (ACE) inhibition on the progression of diabetic nephropathy in rats. Methods: Diabetes (DM) was induced by ip. streptozotocin administration in adult male Sprague-Dawley rats, followed by eight weeks of treatment with the (P)RR blocker „handle region” decoy peptide (HRP, 0,1 mg/kg/day) or with the ACE inhibitor Quinapril (Q, 50 mg/kg/day) and grouped as follows: 1. Control (n=10); 2. DM (n=8); 3. DM+HRP (n=6); 4. DM+Q (n=10); 5. DM+Q+HRP (n=10). Renal functional parameters, histology and gene expressions were evaluated. Results: HRP reduced glomerulosclerosis and podocyte desmin expression, but did not affect proteinuria and tubular ERK(1/2) phosphorylation. Both Q and Q+HRP treatment reduced proteinuria, glomerular and tubular damage, tubular TGF-ß1 expression and ERK(1/2) phosphorylation to the same extent. Conclusion: The effects of HRP were partially beneficial on diabetic kidney lesions as HRP reduced damage but did not improve tubular damage and failed to reduce ERK(1/2) phosphorylation in rats. The combination of HRP with Quinapril had no additive effects over Quinapril monotherapy on the progression of diabetic nephropathy.

Aims We hypothesized that the greater the intra- or interventricular dyssynchrony (intraD, interD), the more effective cardiac resynchronization therapy (CRT) is. We sought to improve patient selection for CRT by using novel ECG dyssynchrony criteria. Methods and results Left ventricular (LV) intraD was estimated by the absolute time difference between the intrinsicoid deflections (ID) in leads aVL and aVF divided by the QRS duration (QRSd): [aVLID − aVFID]/QRSd (%). InterD was estimated from the formula: [ V 5ID − V 1ID]/QRSd (%). Their >25% value indicated electrical dyssynchrony present (ED+) and ≤25% value electrical dyssynchrony absent (ED−) diagnoses. Using the intraD + interD criteria (intra + interDC) together, if at least one of them indicated ED+ diagnosis, a final ED+ diagnosis, if both indicated ED− diagnosis, a final ED− diagnosis was made. Two authors, blinded to CRT response, retrospectively analysed pre-CRT ECGs of 124 patients with known CRT outcome. CRT response was defined as improvement of ≥ 1 NYHA class, being alive and having no hospitalizations for heart failure during 6 months of follow-up. 35/124 (28%) patients were non-responders (NRs), using the traditional criteria (TC) correct diagnosis was made in the remaining 89/124 (72%) responder (R) cases. The test accuracy (TA) of intra + interDC + TC [100/124 (81%), P

Feeding rats with high-fat diet (HFD) with a single streptozotocin (STZ) injection induced obesity, slightly elevated fasting blood glucose and impaired glucose and insulin tolerance, and caused cardiac hypertrophy and mild diastolic dysfunction as published before by Koncsos et al. in 2016. Here we aimed to explore the renal consequences in the same groups of rats. Male Long-Evans rats were fed normal chow (CON, n = 9) or HFD containing 40% lard and were administered STZ at 20 mg/kg (i.p.) at week four (prediabetic rats, PRED, n = 9). At week 21 blood and urine samples were taken and kidney and liver samples were collected for histology, immunohistochemistry and for analysis of gene expression. HFD and STZ increased body weight and visceral adiposity and plasma leptin concentration. Despite hyperleptinemia, plasma C-reactive protein concentration decreased in PRED rats. Immunohistochemistry revealed elevated collagen IV protein expression in the glomeruli, and Lcn2 mRNA expression increased, while Il-1&beta, mRNA expression decreased in both the renal cortex and medulla in PRED vs. CON rats. Kidney histology, urinary protein excretion, plasma creatinine, glomerular Feret diameter, desmin protein expression, and cortical and medullary mRNA expression of TGF-&beta, 1, Nrf2, and PPAR&gamma, were similar in CON and PRED rats. Reduced AMPK&alpha, phosphorylation of the autophagy regulator Akt was the first sign of liver damage, while plasma lipid and liver enzyme concentrations were similar. In conclusion, glomerular collagen deposition and increased lipocalin-2 expression were the early signs of kidney injury, while most biomarkers of inflammation, oxidative stress and fibrosis were negative in the kidneys of obese, prediabetic rats with mild heart and liver injury.

Aim We investigated the influence of aging on the lncRNA profile after renal ischemia-reperfusion (IR) injury in old mice. Methods The left renal pedicle of adult (9.4±0.3 months, n=7) and old (28....

The role of oxidative stress (OXS) due to myocardial nitric oxide synthase (NOS) uncoupling related to oxidative depletion of its cofactor tetrahydrobiopterin (BH4) emerged in the pathogenesis of heart failure with preserved ejection fraction. We determined the prevalence of six single nucleotide polymorphisms (SNPs) of genes encoding enzymes related to OXS, BH4 metabolism, and NOS function in ≥60-year-old 94 patients with hypertension and 18 age-matched controls with normal ejection fraction. Using echocardiography, 56/94 (60%) patients with hypertension had left ventricular (LV) diastolic dysfunction (HTDD+ group) and 38/94 (40%) patients had normal LV diastolic function (HTDD- group). Four SNPs (rs841, rs3783641, rs10483639, and rs807267) of guanosine triphosphate cyclohydrolase-1, the rate-limiting enzyme in BH4 synthesis, one (rs4880) of manganese superoxide dismutase, and one (rs1799983) of endothelial NOS genes were genotyped using real-time polymerase chain reaction method and Taqman probes. Protein carbonylation, BH4, and total biopterin levels were measured from plasma samples. No between-groups difference in minor allele frequency of SNPs was found. We calculated a genetic score indicating risk for OXS based on the minor allele frequencies of the SNPs. A high genetic risk for OXS was significantly associated with HTDD+ even after adjustment for confounding variables (odds ratio [95% confidence interval]:4.79 [1.12-20.54]; P = .035). In both patient groups protein carbonylation (P

(1) Background: Sepsis-induced acute kidney injury (AKI) is the most common form of acute kidney injury (AKI). We studied the temporal profile of the sepsis-induced renal proteome changes. (2) Methods: Male mice were injected intraperitoneally with bacterial lipopolysaccharide (LPS) or saline (control). Renal proteome was studied by LC-MS/MS (ProteomeXchange: PXD014664) at the early phase (EP, 1.5 and 6 h after 40 mg/kg LPS) and the late phase (LP, 24 and 48 h after 10 mg/kg LPS) of LPS-induced AKI. Renal mRNA expression of acute phase proteins (APP) was assessed by qPCR. (3) Results: Renal proteome change was milder in EP vs. LP. APPs dominated the proteome in LP (proteins upregulated at least 4-fold (APPs/all): EP, 1.5 h: 0/10, 6 h: 1/10, LP, 24 h: 22/47, 48 h: 17/44). Lipocalin-2, complement C3, fibrinogen, haptoglobin and hemopexin were the most upregulated APPs. Renal mRNA expression preceded the APP changes with peak effects at 24 h, and indicated renal production of the majority of APPs. (4) Conclusions: Gene expression analysis revealed local production of APPs that commenced a few hours post injection and peaked at 24 h. This is the first demonstration of a massive, complex and coordinated acute phase response of the kidney involving several proteins not identified previously.

MacIver and Townsend's hypothesis predicts, based on a mathematical model of left ventricular contraction, that preserved absolute radial wall thickening (radWT) due to left ventricular hypertrophy is responsible for the normal ejection fraction in patients with heart failure with preserved ejection fraction (HFPEF).We tested the validity of this hypothesis by detailed echocardiography including evaluation of ventricular myocardial strain (S) using speckle tracking imaging in at least 60-year-old 18 controls and 94 hypertensive patients with normal ejection fraction.Echocardiography revealed no left ventricular diastolic dysfunction in 38 out of 94 (40%) patients with hypertension (HTDD-negative group), and 56 out of 94 (60%) patients had diastolic dysfunction (HTDD-positive groups). The absolute values of global longitudinal left ventricular peak systolic S were significantly reduced in both patient groups (P 0.05 for HTDD-negative, P 0.01 for HTDD-positive groups) vs. the controls. There were no significant between-groups differences in circumferential and radial peak left ventricular systolic Ss, radWT and ejection fraction. Left ventricular mass (LVM) (P 0.001), LVM/BMI (P 0.01) increased in the HTDD-positive group and ejection fraction/LVM/BMI decreased in both patient groups (P 0.01 for HTDD-negative, P 0.001 for HTDD-positive groups) vs. the controls. LVM increased, ejection fraction/LVM/BMI decreased in the HTDD-positive group vs. the HTDD-negative group (P 0.05 and P 0.01, respectively).We demonstrated decreased longitudinal left ventricular systolic function and showed that preserved ejection fraction was due to preserved absolute radWT and not due to increased radial or circumferential systolic function in patients with hypertension and normal ejection fraction, a potential HFPEF precursor condition. Instead of ejection fraction, rather ejection fraction/LVM/BMI might be used to detect subtle left ventricular systolic dysfunction in hypertension and HFPEF.

Objectives The diagnostic values of the aVR lead or “Vereckei algorithm,” and the lead II R-wave peak time (RWPT) criterion, recently devised for the differential diagnosis of wide QRS complex tachycardias (WCTs), were compared. Methods A total of 212 WCTs (142 ventricular tachycardias [VTs], 62 supraventricular tachycardias [SVT], and eight preexcitation SVTs) from 145 patients with proven electrophysiologic diagnoses were retrospectively analyzed by seven examiners blinded to the electrophysiologic diagnoses. Results The overall test accuracy of the Vereckei algorithm was superior to that of the RWPT criterion (84.3% vs. 79.6%; p = 0.0003). The sensitivity of the Vereckei algorithm for VT diagnosis was greater than that of RWPT criterion (92.4% vs. 79.1%; p

Complement activation-related pseudoallergy (CARPA) is a frequent side effect of intravenous therapies with nanoparticle-containing drugs and biologicals that are recognized by the immune system as foreign. It is an acute infusion reaction dominated by cutaneous and hemodynamic changes, most significantly a cardiopulmonary distress involving major pulmonary hypertension, systemic hypotension and arrhythmias. Because CARPA is unpredictable by conventional allergy tests and it may be life threatening, it can represent a major barrier to the safe therapeutic application of many modern medicines, including liposomal drugs and monoclonal antibodies. This review summarizes and updates the facts and opens questions regarding this phenomenon, with particular focus on its porcine model.

Preclinical Research The basic hemodynamic, electrophysiological, and pharmacological effects of EGIS-7229 (E-7229) were evaluated and compared with those of a pure “Class III“ antiarrhythmic drug (GLG-V-13) in conscious rabbits. Both compounds decreased heart rate dose-dependently. Mean arterial blood pressure was not influenced by E-7229; however, GLG-V-13 produced a significant elevation on this parameter. Left ventricular end-diastolic pressure was gradually increased by E-7229, while GLG-V-13 induced more considerable elevation on that at intermediate doses. QT and QTcb were lengthened by both compounds; however, higher doses of E-7229 resulted in shortening of the prolonged QT and QTcb. Ventricular effective refractory period (VERP) was significantly prolonged by either drug studied. Threshold doses to produce QT50%, QTmax, VERP50%, and VERPmax were significantly higher for E-7229 compared with GLG-V-13. Significantly higher doses were required for E-7229 to induce arrhythmias. The safety ratio was comparable for both of the compounds. E-7229 can exert multiple actions on cardiac ion channels with minimal influence on hemodynamic parameters and reduced proarrhytmic profile in our preclinical model.

The effects of brain ischemia and reperfusion on smooth muscle function in remote cerebral and peripheral arteries are hardly known. Maximum vasoconstrictions (Emax) caused by 120 mmol/l KCl and 5-HT in endothelium-denuded ring preparations were measured in ischemic and control cerebral arteries of rats after a 1-h right middle cerebral artery occlusion followed by 0-min (I/NR) or 2–3-min (I/SR) reperfusion, and in peripheral arteries after I/SR. Surprisingly, vasoconstrictions to 5-HT and 120 mmol/l K+ were attenuated in remote brain vessels after I/SR, i.e. in the contralateral middle cerebral artery and the basilar artery, while I/NR depressed Emax of 5-HT and high KCl only in the ischemic middle cerebral artery. Pretreatment with N-(2-mercaptopropionyl) glycine (MPG, 100 mg/kg i.p.), a free radical scavenger, fully prevented the impairment of vasomotor function in the middle cerebral artery on both sides after I/SR. Moreover, vasomotor functions were normal in the coronary, renal and pulmonary arteries after I/SR. In conclusion, focal cerebral ischemia and reperfusion impaired vasoconstrictor responses in remote brain arteries of rats by a mechanism involving free radicals. The lack of similar effects in peripheral vessels indicates poor defence of brain arteries against remote injury caused by reactive oxygen species-dependent mechanisms.

Background We recently reported an ECG algorithm for differential diagnosis of regular wide QRS complex tachycardias that was superior to the Brugada algorithm. Objective The purpose of this study was to further simplify the algorithm by omitting the complicated morphologic criteria and restricting the analysis to lead aVR. Methods In this study, 483 wide QRS complex tachycardias [351 ventricular tachycardias (VTs), 112 supraventricular tachycardias (SVTs), 20 preexcited tachycardias] from 313 patients with proven diagnoses were prospectively analyzed by two of the authors blinded to the diagnosis. Lead aVR was analyzed for (1) presence of an initial R wave, (2) width of an initial r or q wave >40 ms, (3) notching on the initial downstroke of a predominantly negative QRS complex, and (4) ventricular activation–velocity ratio (v i /v t ), the vertical excursion (in millivolts) recorded during the initial (v i ) and terminal (v t ) 40 ms of the QRS complex. When any of criteria 1 to 3 was present, VT was diagnosed; when absent, the next criterion was analyzed. In step 4, v i /v t >1 suggested SVT, and v i /v t ≤1 suggested VT. Results The accuracy of the new aVR algorithm and our previous algorithm was superior to that of the Brugada algorithm ( P = .002 and P = .007, respectively). The aVR algorithm and our previous algorithm had greater sensitivity ( P P = .001, respectively) and negative predictive value for diagnosing VT and greater specificity ( P P = .001, respectively) and positive predictive value for diagnosing SVT compared with the Brugada criteria. Conclusion The simplified aVR algorithm classified wide QRS complex tachycardias with the same accuracy as standard criteria and our previous algorithm and was superior to the Brugada algorithm.

The complement (C) activating effect of zymosan and liposomal drugs (AmBisome, Caelyx) leads to significant C consumption in rats, dogs, pigs and other species in vivo, as reflected by a fall in hemolytic complement activity (HCA) of their plasma. However, the acute C activating effect of zymosan and liposomal drugs is unclear in the mouse. Therefore, using sheep red blood cells, we assayed the HCA of plasma obtained from apolipoprotein E-deficient (ApoE) as well as from background C57BL/6 (BL6) mice. Intravenous (i.v.) administration of C activators led to a significant rise (up to 40%) in HCA of the plasma. The HCA steadily rose up to 30 min in ApoE mice, while it peaked at 3 min in BL6 mice, returning to baseline thereafter. The elevated HCA after IV injection of C activators is “paradoxical” in mice, since it implies an increase rather than a decrease in C levels in the blood. One possible explanation of the phenomenon is hemoconcentration due to anaphylatoxin-induced capillary leakage, resulting in an apparent rise of HCA. In conclusion, these preliminary observations highlight, for the first time, a species-dependent opposing impact of C activation and the resulting anaphylatoxin actions on hemolytic complement activity.

To gain insight into the cardiac adaptive mechanisms in diabetes, we studied whether angiotensin II (Ang II) alters expression of the atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and adrenomedullin (AM) genes in the left ventricle of the diabetic rat heart.Diabetes was induced by streptozotocin (STZ; 60 mg/kg body weight intravenously). During the last 24 h of 2.5 or 7 weeks of treatment of male Wistar rats with STZ or vehicle, Ang II (33 microg/kg per h) was administered via osmotic minipumps.Diabetes was associated with an increased left ventricular weight to body weight (LV/BW) ratio, an index of left ventricular hypertrophy, at week 7 but not at week 2.5, and with increased ANP mRNA content at 2.5 weeks, but not with altered expression of the AM and BNP genes. Mean arterial pressure and LV/BW ratio were increased by Ang II in all groups except in the 7-week diabetic group. Levels of ANP mRNA were increased fourfold (P0.001) and threefold (P0.05) by Ang II at 2.5 and 7 weeks in control animals, respectively, and 11-fold (P0.001) and sevenfold (P0.001) at 2.5 and 7 weeks in diabetic animals, respectively. Ang II increased ventricular concentrations of BNP mRNA in control and diabetic animals at 2.5 weeks (1.3-fold, P0.001; and 1.6-fold, P0.001) and at 7 weeks (1.3-fold, P0.05; and 1.8-fold, P0.001), respectively. Left ventricular levels of adrenomedullin mRNA were increased by treatment with Ang II for 24 h in 2.5-week diabetic animals.Ang II markedly increased the levels of natriuretic peptide mRNAs in the left ventricle of normal and diabetic rat hearts, whereas it increased adrenomedullin mRNA levels only in 2.5-week diabetic rats and failed to cause hypertension in 7-week diabetic rats. Left ventricular levels of ANP and BNP mRNA were increased by Ang II in diabetic animals more than the additive effects of diabetes and Ang II alone, showing that Ang II induced an amplified response with respect to cardiac concentrations of ANP and BNP in diabetes.

Oxidative stress and lysosomal phospholipoidosis, which also might be partly attributed to free radicals induced by amiodarone (AM), may be involved in AM toxicity, which can be prevented by antioxidants. Our aim was to study if vitamin E (E) or silymarin (S), a lipid and a water-soluble antioxidant, modified the antiarrhythmic efficacy of AM in a rat reperfusion arrhythmia test. The following groups of male Sprague-Dawley rats (15 rats/group) were treated by gavage once a day for 4 weeks: 1. methylcellulose (MC, 0.4%), 2. sunflower seed oil (SSO), 3. AM, suspended in MC (30 mg/kg), 4. E, dissolved in SSO (100 mg/kg), 5. AM + E, 6. S, suspended in MC (80 mg/kg), 7. AM + S. The mean duration of ventricular tachycardia + fibrillation (MDVT + VF) and sinus rhythm (MDSR) the incidence of ventricular fibrillation (VF) and ventricular tachycardia (VT) and mortality were measured during a 10-min reperfusion after a 5-min coronary artery occlusion in anaesthetized rats. An arrhythmia score, representing the combined incidence and duration of different types of ventricular arrhythmia, was calculated. Compared with the MC group, MDSR was longer and MDVT + VF was shorter in all drug treated groups and in the SSO group. In the AM + E treated group MDSR was prolonged more and MDVT + VF was shortened more than in the AM, E or SSO groups. Compared with the MC group, the incidence of VF and mortality was similarly decreased in the SSO group and in most drug treated groups. No significant difference in the incidence of VT was found among all groups. The arrhythmia score was reduced by all drug treatments. Combined treatment with AM + E decreased arrhythmia score more than treatment with AM or SSO alone, but arrhythmia score was similar in the AM + E and E groups. In conclusion, both AM and antioxidant treatments alone or together resulted in a marked reduction of reperfusion arrhythmias in this model. SSO also exerted a moderate antiarrhythmic effect. Antioxidants administered together with AM did not attenuate and E might have even enhanced the antiarrhythmic effect of AM, therefore the combination of antioxidants with AM may be advantageous to reduce AM toxicity.

EGIS-7229 is a novel antiarrhythmic candidate having multiple mechanisms of action with class III predominance. In this study, the effects of EGIS-7229 and sotalol on action potential duration (APD) and incidence of early afterdepolarizations (EADs) were studied and compared in rabbit papillary muscle by using conventional microelectrode techniques. In control bathing solution, both drugs increased APD in a concentration-dependent manner; however, the prolongation of APD was greater with sotalol than with EGIS-7229 when the same drug concentrations were compared. EAD developed in 3 of the 11 preparations (27%) bathed with a solution containing 3.6 mmol/l CsCl + 2 mmol/l KCl within the first 120 min of superfusion. The addition of 100 μmol/l sotalol to this superfusate increased the incidence of EAD to 83% (10 from 12), whereas the addition of the same concentration of EGIS-7229 prevented the development of EAD in all of the 9 preparations studied. These differences in incidence of EAD are likely attributable to differences in drug-induced increases of APD-50 in the presence of CsCl. Prolongation of APD-90 showed less correlation with incidence of EAD than changes in APD-50. On the basis of these in vitro results, high concentrations of EGIS-7229 cannot be expected to be torsadogenic in vivo—in contrast with sotalol—presumably owing to the combined class III + IV activity of the compound.

Background Detection of acute kidney injury (AKI) is still a challenge if conventional markers of kidney function are within reference range. We studied the sensitivity and specificity of NGAL as an AKI marker at different degrees of renal ischemia. Methods Male C57BL/6J mice were subjected to 10-, 20- or 30-min unilateral renal ischemia, to control operation or no operation, and AKI was evaluated 1 day later by histology, immunohistochemistry, BUN, creatinine, NGAL (plasma and urine) and renal NGAL mRNA expression. Results A short (10-min) ischemia did not alter BUN or kidney histology, but elevated plasma and urinary NGAL level and renal NGAL mRNA expression although to a much smaller extent than longer ischemia. Surprisingly, control operation elevated plasma NGAL and renal NGAL mRNA expression to a similar extent as 10-min ischemia. Further, the ratio of urine to plasma NGAL was the best parameter to differentiate a 10-min ischemic injury from control operation, while it was similar in the non and control-operated groups. Conclusions These results suggest that urinary NGAL excretion and especially ratio of urine to plasma NGAL are sensitive and specific markers of subclinical acute kidney injury in mice.

Bromoethylamine (BEA, 30-40 mg/kg) was administered to dogs to determine whether damage to the inner medulla of the kidney, the putative source of a depressor hormone, causes hypertension in this species. Bromoethylamine produces hypertension in rats but this has not been confirmed in other species, although we have shown that this dose of BEA in dogs abolishes the release of a reno-medullary vasodepressor hormone in response to marked increases in renal perfusion pressure. During acute BEA administration over 1 h to conscious dogs, there were no significant effects on renal blood flow, arterial pressure or total peripheral resistance, but there was a significantly greater diuresis compared to vehicle administration. Over the first 10-14 days after BEA, daily urine output rose 5-10 fold initially and plasma creatinine concentration rose markedly. There was no significant effect on arterial pressure, cardiac output, total peripheral resistance, or renal blood flow over this period. BEA administration caused extensive damage to the thin limbs of the loops of Henle, widespread thrombosis of blood vessels and haemorrhage into the interstitium of the dog renal medulla. Reno-medullary interstitial cells were devoid of lipid droplets, were synthetic, and were associated with increased amounts of extracellular matrix. Thus extensive renal medullary damage by BEA administration to conscious dogs did not alter resting systemic haemodynamics, and these results therefore provide no evidence for a role for the medulla in the maintenance of resting arterial pressure in the dog.

Aims The Brugada criteria proposed to distinguish between regular, monomorphic wide QRS complex tachycardias (WCT) caused by supraventricular (SVT) and ventricular tachycardia (VT) have been reported to have a better sensitivity and specificity than the traditional criteria. By incorporating two new criteria, a new, simplified algorithm was devised and compared with the Brugada criteria. Methods and results A total of 453 WCTs (331 VTs, 105 SVTs, 17 pre-excited tachycardias) from 287 consecutive patients with a proven electrophysiological (EP) diagnosis were prospectively analysed by two of the authors blinded to the EP diagnosis. The following criteria were analysed: (i) presence of AV dissociation; (ii) presence of an initial R wave in lead aVR; (iii) whether the morphology of the WCT correspond to bundle branch or fascicular block; (iv) estimation of initial ( v i) and terminal ( v t) ventricular activation velocity ratio ( v i/ v t) by measuring the voltage change on the ECG tracing during the initial 40 ms ( v i) and the terminal 40 ms ( v t) of the same bi- or multiphasic QRS complex. A v i/ v t >1 was suggestive of SVT and a v i/ v t ≤1 of VT. An initial R wave in lead aVR suggested VT. The overall test accuracy of the new algorithm was superior ( P = 0.006) to that of the Brugada criteria. The new algorithm had a greater sensitivity ( P < 0.001) and (−) predictive value (NPV) for VT diagnosis, and specificity ( P = 0.0471) and (+) predictive value (PPV) for SVT diagnosis than those of the Brugada criteria [both NPV for VT diagnosis and PPV for SVT diagnosis were: 83.5% (95% confidence interval = CI 75.9–91.1%) for the new vs. 65.2% (95% CI 56.5–73.9%) for the Brugada algorithms]. Conclusion The new algorithm is a highly accurate tool for correctly diagnosing the cause of WCT ECGs.

Excessive stimulation of N-methyl-D-aspartate (NMDA) receptors during ischemia contributes to apoptotic and excitotoxic nerve cell death. Kynurenic acid is a selective antagonist at the glycine co-agonist site of the NMDA receptor complex at low concentration, and it is a broad-spectrum excitatory amino acid receptor blocker at high concentration. Kynurenic acid provides neuroprotection in animal models of cerebral ischemia only at very high doses as it hardly crosses the blood-brain barrier. The neuroprotective effect of L-kynurenine sulfate, a precursor of kynurenic acid, was therefore studied because L-kynurenine readily crosses the blood-brain barrier. L-kynurenine sulfate was administered 15 min before permanent focal cerebral ischemia produced by electrocoagulation of the distal middle cerebral artery in mice. L-kynurenine sulfate induced a small decrease in the surface area of the brain infarction (10%, P

EGIS-8332 and GYKI 53405 are selective, non-competitive AMPA (2-amino-3[3-hydroxy-5-methyl-4-isoxazolyl] propionic acid) antagonists that effectively protected against tissue injury caused by global and focal cerebral ischemia in laboratory animals. This study evaluated the therapeutic time window of neuroprotection by EGIS-8332 and GYKI 53405 in permanent and transient middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. Infarct size was measured by TTC staining 48 h after permanent MCAO (electrocoagulation), and 24 h after reperfusion following a 1-h transient MCAO carried out using the intraluminal filament technique. Treatment with EGIS-8332 (10 mg/kg, i.p.) 60 or 120 min after permanent MCAO, decreased infarct size by 30% and 36%, respectively, and the effect of GYKI 53405 (10 mg/kg, i.p.) was similar (30% and 33%, respectively; p

2,3,5-Triphenyltetrazolium chloride (TTC) staining measures tissue viability used to evaluate infarct size. The goal of this study was to compare viability of neuronal tissue during the early phases of ischemia-reperfusion assessed either by perfusion of the brain with TTC solution transcardially, in vivo, or by staining brain slices, in vitro.The middle cerebral artery was occluded for 1 h in male SPRD rats and then reperfused for 0, 1, 4, 8, 16 and 24 h. Ischemic damage was evaluated by TTC staining, in vivo and in vitro, and by histology (Luxol Fast Blue and Fluoro-Jade staining, electron microscopy).Core volume of tissue injury measured in vivo was large at 0 h and steadily decreased by 50% (p0.001) up to 16 h, but substantially increased from 16 to 24 h of reperfusion. In contrast, a significant core volume appeared at 4 h only, in vitro, and gradually increased up to 24 h. Core volume was larger in vivo than in vitro at all times except at 16 h when the opposite was observed. Evans blue administered intracardially stained TTC-negative areas at 1 and 24 h. Histology covered the evolution of serious tissue injury but also demonstrated some morphologically preserved neurons in the infracted area at 24 h.Formation of formazan from TTC can depend on both the staining method and the metabolic burden of the brain tissue causing uncertainties in the volume of ischemia-induced brain injury measured by TTC staining.

The I(Kr) blocker EGIS-7229 (S-21407), displays class Ib and class IV effects that may alter its pharmacologic profile compared with those of pure I(Kr) blockers. Therefore, the concentration- and frequency-dependent effects of EGIS-7229, and of the I(Kr) blockers d,l-sotalol and dofetilide, on the effective refractory period (ERP) were measured in isolated right ventricular papillary muscle of the rabbit in vitro. The effects of these drugs on right ventricular fibrillation threshold (RVFT) at increasing intravenous doses were also determined in anesthetized cats. Dofetilide and d,l-sotalol increased ERP in a concentration-dependent manner (dofetilide: 3-100 nM; d,l-sotalol: 3-100 microM) with strong reverse frequency dependence at high concentrations. EGIS-7229 concentration dependently lengthened ERP at 1-30 microM. Its effect on ERP was clearly reverse frequency dependent at 3 microM, but this feature of the drug diminished at 10 microM and was not apparent at 30 microM. The effect of EGIS-7229 (30 microM) on ERP was devoid of reverse frequency dependence as it was more effective (31%) than dofetilide (16 %) at high-pacing rate (3 Hz), whereas it was less effective (50%) than dofetilide (70%) at slow-pacing rate (1 Hz). Reverse frequency-dependent ERP effect of dofetilide (100 nM) was similarly abolished by the addition of lidocaine (30 microM). EGIS-7229 (1-8 mg/kg iv), d,l-sotalol (1-8 mg/kg iv), and dofetilide (10-80 microg/kg iv) caused a dose-dependent increase in RVFT. The minimum effective dose of d,l-sotalol and EGIS-7229 was 1 and 2 mg/kg, respectively, whereas that of dofetilide was 10 microg/kg. EGIS-7229 induced a smaller peak effect in RVFT than sotalol or dofetilide. In conclusion, EGIS-7229 markedly increased refractoriness to electrical stimulation in vitro and in vivo. Compared with pure I(Kr) blockers, the benefits of EGIS-7229 seem to be a greater lengthening of effective refractory period at rapid stimulation rates, suggesting a strong antiarrhythmic action, and a smaller effect at slow stimulation rates, suggesting low potential to induce early afterdepolarizations.

The aim of the study was to examine the possible involvement of cholecystokinin (by lorglumide) and cholinergic mechanisms (by atropine) in magnesium sulphate (MgSO(4))-induced gallbladder contraction of conscious dogs. The gallbladder (GB) volume was determined by ultrasonography. The optimal dose of 80 mg kg(-1)of MgSO(4)was determined from a MgSO(4)dose-response curve using doses of 10, 20,40, 80, 120 mg kg(-1). The largest dose of MgSO(4)was less effective than the optimal dose. Peak gallbladder contraction (32 per cent) was achieved at 30 minutes. Atropine (50 microg kg(-1)s.c.) or lorglumide (1 mg kg(-1)p.o.) fully prevented GB contraction. In conclusion, supraoptimal doses of MgSO(4)have a diminishing effect. The sustained contraction of the gallbladder in response to the optimal dose of MgSO(4)can be explained by an additive effect of the cholecystokinin release and a cholinergic trigger mechanism. Ultrasonography and MgSO(4)stimulation proved to be a valuable technique for examination of gallbladder motility.

Acute ischemic renal failure is of great clinical importance because of its frequent occurrence and the high mortality it causes. Recent observations indicate that reperfusion has its own dangers because of oxygen-derived free radicals. To study this problem, ischemia was evoked in dogs in one kidney, by clamping the left renal artery for 45 min. This was followed by a 90-min period of reperfusion when diuresis, GFR, PAH clearance and sodium and potassium excretion were studied. Besides a control group (n = 6), the following treatment groups were investigated. Allopurinol (n = 7): 50 mg/kg for two days p.o. and 50 mg/kg in physiological saline infusion during the experiment; a small dose of SOD (n = 6): 0.5 mg/kg in infusion, started 1 min before reperfusion and given continuously for 10 min; and a high dose of SOD (n = 7): 5 mg/kg as above. In the first 15 min following reperfusion, the renal functions significantly worsened in all groups. Later on, the renal functions gradually improved and in the last period after reperfusion, GFR in the ischemic kidney was 64%, cPAH 59%, diuresis 60% and sodium and potassium excretion were 65% and 76%, respectively, of the basal values in the control group. Treatment with free radical scavengers did not cause any considerable changes in the renal functions. In some respects, the worst results were observed with low-level SOD treatment (cPAH, diuresis, as well as sodium and potassium excretion). At the end of reperfusion, there was a significant drop in sodium excretion by the right (intact circulation) kidney of the treated animals.