Maximilia Hottenrott

N-octanoyl-Dopamine Is an Agonist at the Capsaicin Receptor TRPV1 and Mitigates Is Chemia-Induced Acute Kidney Injury in Rat

Since stimulation of transient receptor potential channels of the vanilloid receptor subtype 1 (TRPV1) mitigates acute kidney injury (AKI) and endogenous N-acyl dopamine derivatives are able to activate TRPV1, we tested if synthetic N-octanoyl-dopamine (NOD) activates TRPV1 and if it improves AKI. These properties of NOD and its intrinsic anti-inflammatory character were compared with those of dopamine (DA). TRPV1 activation and anti-inflammatory properties of NOD and DA were tested using primary cell cultures in vitro. The influence of NOD and DA on AKI was tested in a prospective, randomized, controlled animal study with 42 inbred male Lewis rats (LEW, RT1), treated intravenously with equimolar concentrations of DA or NOD one hour before the onset of warm ischemia and immediately before clamp release. NOD, but not DA, activates TRPV1 channels in isolated dorsal root ganglion neurons (DRG) that innervate several tissues including kidney. In TNFα stimulated proximal tubular epithelial cells, inhibition of NFκB and subsequent inhibition of VCAM1 expression by NOD was significantly stronger than by DA. NOD improved renal function compared to DA and saline controls. Histology revealed protective effects of NOD on tubular epithelium at day 5 and a reduced number of monocytes in renal tissue of DA and NOD treated rats. Our data demonstrate that NOD but not DA activates TRPV1 and that NOD has superior anti-inflammatory properties in vitro. Although NOD mitigates deterioration in renal function after AKI, further studies are required to assess to what extend this is causally related to TRPV1 activation and/or desensitization.

End-ischemic machine perfusion reduces bile duct injury in donation after circulatory death rat donor livers independent of the machine perfusion temperature

A short period of oxygenated machine perfusion (MP) after static cold storage (SCS) may reduce biliary injury in donation after cardiac death (DCD) donor livers. However, the ideal perfusion temperature for protection of the bile ducts is unknown. In this study, the optimal perfusion temperature for protection of the bile ducts was assessed. DCD rat livers were preserved by SCS for 6 hours. Thereafter, 1 hour of oxygenated MP was performed using either hypothermic machine perfusion, subnormothermic machine perfusion, or with controlled oxygenated rewarming (COR) conditions. Subsequently, graft and bile duct viability were assessed during 2 hours of normothermic ex situ reperfusion. In the MP study groups, lower levels of transaminases, lactate dehydrogenase (LDH), and thiobarbituric acid reactive substances were measured compared to SCS. In parallel, mitochondrial oxygen consumption and adenosine triphosphate (ATP) production were significantly higher in the MP groups. Biomarkers of biliary function, including bile production, biliary bicarbonate concentration, and pH, were significantly higher in the MP groups, whereas biomarkers of biliary epithelial injury (biliary gamma‐glutamyltransferase [GGT] and LDH), were significantly lower in MP preserved livers. Histological analysis revealed less injury of large bile duct epithelium in the MP groups compared to SCS. In conclusion, compared to SCS, end‐ischemic oxygenated MP of DCD livers provides better preservation of biliary epithelial function and morphology, independent of the temperature at which MP is performed. End‐ischemic oxygenated MP could reduce biliary injury after DCD liver transplantation. Liver Transpl 21:1300‐1311, 2015.

Dopamine and Lipophilic Derivates Protect Cardiomyocytes against Cold Preservation Injury

Donor heart allografts are extremely susceptible to prolonged static cold storage. Because donor treatment with low-dose dopamine improves clinical outcome after heart transplantation, we tested the hypothesis that dopamine and its lipophilic derivate, N-octanoyl dopamine (NOD), protect cardiomyocytes from cold storage injury. Neonatal rat cardiomyocytes were treated with dopamine or NOD or left untreated and subsequently subjected to static cold storage (8–12 hours). Dopamine- and NOD-treated cardiomyocytes displayed a better viability compared with untreated cells after hypothermia. In untreated cardiomyocytes, cell damage was reflected by lactate dehydrogenase (LDH) release and a decrease in intracellular ATP. NOD was approximately 20-fold more potent than dopamine. Similarly to cardiomyocytes in vitro, rat hearts perfused with NOD before explantation showed significantly lower LDH release after static cold storage. ATP regeneration and spontaneous contractions after cold storage and rewarming only occurred in treated cardiomyocytes. Hypothermia severely attenuated isoprenaline-induced cAMP formation in control but not in dopamine- or NOD-treated cells. Esterified derivates of NOD with redox potential and lipophilic side chains reduced cell damage during cold storage similarly to NOD. In contrast to dopamine, neither NOD nor its derivates induced a significant β-adrenoceptor–mediated elevation of cellular cAMP levels. The β1-adrenoceptor antagonist atenolol and D1/D2 receptor antagonist fluphenazine had no impact on the protective effect of NOD or dopamine. We conclude that dopamine as well as NOD treatment mitigates cold preservation injury to cardiomyocytes. The beneficial effects are independent of β-adrenoceptor or dopaminergic receptor stimulation but correlate with redox potential and lipophilic properties.

Open lung approach with low tidal volume mechanical ventilation attenuates lung injury in rats with massive brain damage.

IntroductionThe ideal ventilation strategy for patients with massive brain damage requires better elucidation. We hypothesized that in the presence of massive brain injury, a ventilation strategy using low (6 milliliters per kilogram ideal body weight) tidal volume (VT) ventilation with open lung positive end-expiratory pressure (LVT/OLPEEP) set according to the minimal static elastance of the respiratory system, attenuates the impact of massive brain damage on gas-exchange, respiratory mechanics, lung histology and whole genome alterations compared with high (12 milliliters per kilogram ideal body weight) VT and low positive end-expiratory pressure ventilation (HVT/LPEEP).MethodsIn total, 28 adult male Wistar rats were randomly assigned to one of four groups: 1) no brain damage (NBD) with LVT/OLPEEP; 2) NBD with HVT/LPEEP; 3) brain damage (BD) with LVT/OLPEEP; and 4) BD with HVT/LPEEP. All animals were mechanically ventilated for six hours. Brain damage was induced by an inflated balloon catheter into the epidural space. Hemodynamics was recorded and blood gas analysis was performed hourly. At the end of the experiment, respiratory system mechanics and lung histology were analyzed. Genome wide gene expression profiling and subsequent confirmatory quantitative polymerase chain reaction (qPCR) for selected genes were performed.ResultsIn NBD, both LVT/OLPEEP and HVT/LPEEP did not affect arterial blood gases, as well as whole genome expression changes and real-time qPCR. In BD, LVT/OLPEEP, compared to HVT/LPEEP, improved oxygenation, reduced lung damage according to histology, genome analysis and real-time qPCR with decreased interleukin 6 (IL-6), cytokine-induced neutrophil chemoattractant 1 (CINC)-1 and angiopoietin-4 expressions. LVT/OLPEEP compared to HVT/LPEEP improved overall survival.ConclusionsIn BD, LVT/OLPEEP minimizes lung morpho-functional changes and inflammation compared to HVT/LPEEP.

N-Octanoyl Dopamine Inhibits the Expression of a Subset of κB Regulated Genes: Potential Role of p65 Ser276 Phosphorylation

Background and Purpose Catechol containing compounds have anti-inflammatory properties, yet for catecholamines these properties are modest. Since we have previously demonstrated that the synthetic dopamine derivative N-octanoyl dopamine (NOD) has superior anti-inflammatory properties compared to dopamine, we tested NOD in more detail and sought to elucidate the molecular entities and underlying mechanism by which NOD down-regulates inflammation. Experimental Approach Genome wide gene expression profiling of human umbilical vein endothelial cells (HUVECs) was performed after stimulation with TNF-α or in the combination with NOD. Confirmation of these differences, NFκB activation and the molecular entities that were required for the anti-inflammatory properties were assessed in subsequent experiments. Key Results Down regulation of inflammatory genes by NOD occurred predominantly for κB regulated genes, however not all κB regulated genes were affected. These findings were explained by inhibition of RelA phosphorylation at Ser276. Leukocyte adherence to TNF-α stimulated HUVECs was inhibited by NOD and was reflected by a diminished expression of adhesion molecules on HUVECs. NOD induced HO-1 expression, but this was not required for inhibition of NFκB. The anti-inflammatory effect of NOD seems to involve the redox active catechol structure, although the redox active para-dihydroxy benzene containing compounds also displayed anti-inflammatory effects, provided that they were sufficiently hydrophobic. Conclusions and Implications The present study highlighted important mechanisms and molecular entities by which dihydroxy benzene compounds exert their potential anti-inflammatory action. Since NOD does not have hemodynamic properties, NOD seems to be a promising candidate drug for the treatment of inflammatory diseases.

Inadequate Antioxidative Responses in Kidneys of Brain-Dead Rats

Background Brain death (BD)-related lipid peroxidation, measured as serum malondialdehyde (MDA) levels, correlates with delayed graft function in renal transplant recipients. How BD affects lipid peroxidation is not known. The extent of BD-induced organ damage is influenced by the speed at which intracranial pressure increases. To determine possible underlying causes of lipid peroxidation, we investigated the renal redox balance by assessing oxidative and antioxidative processes in kidneys of brain-dead rats after fast and slow BD induction. Methods Brain death was induced in 64 ventilated male Fisher rats by inflating a 4.0F Fogarty catheter in the epidural space. Fast and slow inductions were achieved by an inflation speed of 0.45 and 0.015 mL/min, respectively, until BD confirmation. Healthy non–brain-dead rats served as reference values. Brain-dead rats were monitored for 0.5, 1, 2, or 4 hours, after which organs and blood were collected. Results Increased MDA levels became evident at 2 hours of slow BD induction at which increased superoxide levels, decreased glutathione peroxidase (GPx) activity, decreased glutathione levels, increased inducible nitric oxide synthase and heme-oxygenase 1 expression, and increased plasma creatinine levels were evident. At 4 hours after slow BD induction, superoxide, MDA, and plasma creatinine levels increased further, whereas GPx activity remained decreased. Increased MDA and plasma creatinine levels also became evident after 4 hours fast BD induction. Conclusion Brain death leads to increased superoxide production, decreased GPx activity, decreased glutathione levels, increased inducible nitric oxide synthase and heme-oxygenase 1 expression, and increased MDA and plasma creatinine levels. These effects were more pronounced after slow BD induction. Modulation of these processes could lead to decreased incidence of delayed graft function.

N-octanoyl Dopamine Attenuates the Development of Transplant Vasculopathy in Rat Aortic Allografts Via Smooth Muscle Cell Protective Mechanisms

Background Transplant vasculopathy (TV) is a major cause for late graft loss after cardiac transplantation. Endothelial damage and T cell infiltration play a pivotal role in the development of TV. Because N-octanoyl dopamine (NOD) inhibits vascular inflammation and suppresses T cell activation in vitro, we here tested the hypothesis that NOD treatment ameliorates TV. Methods Aortic grafts were orthotopically transplanted in the Dark Agouti to Brown Norway strain combination. Recipient rats were treated with NOD or vehicle administered via osmotic minipumps. Histology and quantitative polymerase chain reaction (qPCR) were performed on nontransplanted aortas and grafts explanted 2 and 4 weeks after transplantation to assess the degree of TV, inflammation, apoptosis, and number of (proliferating) &agr; smooth muscle actin (&agr;SMA)+ neointimal cells. In vitro analyses of human aortic smooth muscle cells were performed to test the effect of NOD on proliferation (WST-1 assay), cell cycle (flow cytometry and qPCR), and cytokine-induced apoptosis (flow cytometry). Results Allografts from vehicle-treated recipients developed neointimal lesions predominantly consisting of &agr;SMA-expressing cells. NOD treatment significantly reduced neointima formation and neointimal &agr;SMA+ cells. In situ, smooth muscle cell proliferation (Ki67) was not influenced by NOD. Macrophage (CD68), T (CD3), and Natural Killer (ANK61) cell infiltration as well as intragraft TNF&agr; and IFN&ggr; mRNA expression were similar in both groups. Medial apoptosis (cleaved caspase-3) was significantly reduced by NOD. In vitro, NOD inhibited proliferation of human aortic smooth muscle cells by causing a G1-arrest and protected from TNF&agr;-induced apoptosis. Conclusions This study identified NOD as potential treatment modality to attenuate TV. Our data clearly support a vasculoprotective effect of NOD by reducing smooth muscle cell proliferation and inflammation-induced apoptosis.

Effects of mechanical ventilation on gene expression profiles in renal allografts from brain dead rats

Pathophysiological changes of brain death (BD) are impairing distal organ function and harming potential renal allografts. Whether ventilation strategies influence the quality of renal allografts from BD donors has not been thoroughly studied. 28 adult male Wistar rats were randomly assigned to four groups: 1) no brain death (NBD) with low tidal volume/low positive endexpiratory pressure (PEEP) titrated to minimal static elastance of the respiratory system (LVT/OLPEEP); 2) NBD with high tidal volume/low PEEP (HVT/LPEEP); 3) brain death (BD) with LVT/OLPEEP; and 4) BD with HVT/LPEEP. We hypothesized that HVT/LPEEP in BD leads to increased interleukin 6 (IL-6) gene expression and impairs potential renal allografts after six hours of mechanical ventilation. We assessed inflammatory cytokines in serum, genome wide gene expression profiles and quantitative PCR (qPCR) in kidney tissue. The influence of BD on renal gene-expression profiles was greater than the influence of the ventilation strategy. In BD, LVT ventilation did not influence the inflammatory parameters or kidney function in our experimental model.