TY  - JOUR
A1  - Palkovits, Miklós
A1  - Šebeková, Katarína
A1  - Klenovics, Kristina Simon
A1  - Kebis, Anton
A1  - Fazeli, Gholamreza
A1  - Bahner, Udo
A1  - Heidland, August
T1  - Neuronal Activation in the Central Nervous System of Rats in the Initial Stage of Chronic Kidney Disease-Modulatory Effects of Losartan and Moxonidine
JF  - PLoS ONE
N2  - The effect of mild chronic renal failure (CRF) induced by 4/6-nephrectomy (4/6NX) on central neuronal activations was investigated by c-Fos immunohistochemistry staining and compared to sham-operated rats. In the 4/6 NX rats also the effect of the angiotensin receptor blocker, losartan, and the central sympatholyticum moxonidine was studied for two months. In serial brain sections Fos-immunoreactive neurons were localized and classified semiquantitatively. In 37 brain areas/nuclei several neurons with different functional properties were strongly affected in 4/6NX. It elicited a moderate to high Fos-activity in areas responsible for the monoaminergic innervation of the cerebral cortex, the limbic system, the thalamus and hypothalamus (e.g. noradrenergic neurons of the locus coeruleus, serotonergic neurons in dorsal raphe, histaminergic neurons in the tuberomamillary nucleus). Other monoaminergic cell groups (A5 noradrenaline, C1 adrenaline, medullary raphe serotonin neurons) and neurons in the hypothalamic paraventricular nucleus (innervating the sympathetic preganglionic neurons and affecting the peripheral sympathetic outflow) did not show Fos-activity. Stress- and pain-sensitive cortical/subcortical areas, neurons in the limbic system, the hypothalamus and the circumventricular organs were also affected by 4/6NX. Administration of losartan and more strongly moxonidine modulated most effects and particularly inhibited Fos-activity in locus coeruleus neurons. In conclusion, 4/6NX elicits high activity in central sympathetic, stress- and pain-related brain areas as well as in the limbic system, which can be ameliorated by losartan and particularly by moxonidine. These changes indicate a high sensitivity of CNS in initial stages of CKD which could be causative in clinical disturbances.
KW  - brain natriuretic peptide
KW  - kidneys
KW  - cognitive impairment
KW  - central nervous system
KW  - chronic kidney disease
KW  - neurons
KW  - homeostasis
KW  - blood pressure
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130108
VL  - 8
IS  - 6
ER  - 
TY  - THES
A1  - Fazeli, Gholamreza
T1  - Signaling in the induction of genomic damage by endogenous compounds
T1  - Signalwege bei der Induktion von Genomschäden durch endogene Substanzen
N2  - Reactive oxygen species (ROS) are continuously generated in cells and are involved in physiological processes including signal transduction but also their damaging effects on biological molecules have been well described. A number of reports in the literature implicate excessive oxidative stress and/or inadequate antioxidant defense in the pathogenesis of cancer, atherosclerosis, chronic and age related disorders. Several studies have indicated that activation of the renin-angiotensin-aldosterone-system can lead to the formation of ROS. Epidemiological studies have revealed higher renal cell cancer incidences and also higher cancer mortalities in hypertensive individuals. Recently, our group has shown that perfusion of the isolated mouse kidney with Ang II or treatment of several cell lines with Ang II leads to formation of DNA damage and oxidative base modifications. Here, we tried to scrutinize the pathway involved in genotoxicity of Ang II. We confirmed the genotoxicity of Ang II in two kidney cell lines of human origin. Ang II treatment led to the production of superoxide anions which we could hinder when we used the membrane permeable superoxide dismutase (SOD) mimetic TEMPOL. One of the enzymes which is activated in the cells after Ang II treatment and is able to produce ROS is NADPH oxidase. We demonstrated the activation of NADPH oxidase in response to Ang II by upregulation of its p47 subunit using RT-PCR. Also, pPhosphorylation of p47 subunit of NADPH oxidase after Ang II treatment was enhanced. Using two inhibitors we showed that NADPH oxidase inhibition completely prevents DNA damage by Ang II treatment. To differentiate between Nox2 and Nox4 isoforms of NADPH oxidase subunits in the genotoxicity of Ang II, we performed siRNA inhibition and found a role only for Nox4, while Nox2 was not involved. Next, we investigated PKC as a potential activator of NADPH oxidase. We showed that PKC becomes phosphorylated after Ang II treatment and also that inhibition of PKC hinders Ang II from damaging the cells. Our results from using several inhibitors of different parts of the pathway revealed that PKC activation in this pathway is dependent on the action of PLC on membrane phospholipids and production of IP3. IP3 binds to its receptor at endoplasmic reticulum (ER), opening a channel which allows calcium efflux into the cytoplasm. In this manner, both ER calcium stores and extracellular calcium cooperate so that Ang II can exert its genotoxic effect. PLC is activated by AT1R stimulation. We could also show that the genotoxicity of Ang II is mediated via AT1R signaling using the AT1R antagonist candesartan. In conclusion, here we have shown that Ang II is able to damage genomic damage in cell lines of kidney origin. The observed damage is associated with production of ROS. A decrease in Ang II-induced DNA damage was observed after inhibition of G-proteins, PLC, PKC and NADPH oxidase and interfering with intra- as well as extracellular calcium signaling. This leads to the following preliminary model of signaling in Ang II-induced DNA damage: binding of Ang II to the AT1 receptor activates PLC via stimulation of G-proteins, resulting in the activation of PKC in a calcium dependent manner which in turn, activates NADPH oxidase. NADPH oxidase with involvement of its Nox4 subunit then produces reactive oxygen species which cause DNA damage. Dopamine content and metabolism in the peripheral lymphocytes of PD patients are influenced by L-Dopa administration. The PD patients receiving a high dose of L-Dopa show a significantly higher content of dopamine in their lymphocytes compared to PD patients who received a low dose of L-Dopa or the healthy control. Central to many of the processes involved in oxidative stress and oxidative damage in PD are the actions of monoamine oxidase (MAO), the enzyme which is responsible for the enzymatic oxidation of dopamine which leadsing to production of H2O2 as a by-product. We investigated whether dopamine oxidation can cause genotoxicity in lymphocytes of PD patents who were under high dose L-Dopa therapy and afterward questioned the occurrence of DNA damage after dopamine treatment in vitro and tried to reveal the mechanism by which dopamine exerts its genotoxic effect. The frequency of micronuclei in peripheral blood lymphocytes of the PD patients was not elevated compared to healthy age-matched individuals, although the formation of micronuclei revealed a positive correlation with the daily dose of L-Dopa administration in patients who received L-Dopa therapy together with dopamine receptor agonists. In vitro, we describe an induction of genomic damage detected as micronucleus formation by low micromolar concentrations in cell lines with of different tissue origins. The genotoxic effect of dopamine was reduced by addition of the antioxidants TEMPOL and dimethylthiourea which proved the involvement of ROS production in dopamine-induced DNA damage. To determine whether oxidation of dopamine by MAO is relevant in its genotoxicity, we inhibited MAO with two inhibitors, trans-2-phenylcyclopropylamine hydrochloride (PCPA) and Ro 16-6491 which both reduced the formation of micronuclei in PC-12 cells. We also studied the role of the dopamine transporter (DAT) and dopamine type 2 receptor (D2R) signaling in the genotoxicity of dopamine. Inhibitors of the DAT, GBR-12909 and nomifensine, hindered dopamine-induced genotoxicity. These results were confirmed by treatment of MDCK and MDCK-DAT cells, the latter containing the human DAT gene, with dopamine. Only MDCK-DAT cells showed elevated chromosomal damage and dopamine uptake. Although stimulation of D2R with quinpirole in the absence of dopamine did not induce genotoxicity in PC-12 cells, interference with D2R signaling using D2R antagonist and inhibition of G-proteins, phosphoinositide 3 kinase and extracellular signal-regulated kinases reduced dopamine-induced genotoxicity and affected the ability of DAT to take up dopamine. Furthermore, the D2R antagonist sulpiride inhibited the dopamine-induced migration of DAT from cytosol to cell membrane. Overall, the neurotransmitter dopamine causes DNA damage and oxidative stress in vitro. There are also indications that high dose L-Dopa therapy might lead to oxidative stress. Dopamine exerts its genotoxicity in vitro upon transport into the cells and oxidization oxidation by MAO. Transport of dopamine by DAT has the central role in this process. D2R signaling is involved in the genotoxicity of dopamine by affecting activation and cell surface expression of DAT and hence modulating dopamine uptake. We provided evidences for receptor-mediated genotoxicity of two compounds with different mechanism of actions. The involvement of these receptors in many human complications urges more investigations to reveal whether abnormalities in the endogenous compounds-mediated signaling can play a role in the initiation of new conditions like carcinogenesis.
N2  - Reaktive Sauerstoffspezies (ROS) werden kontinuierlich in Zellen generiert und sind an physiologischen Prozessen wie der Signaltransduktion beteiligt. Aber auch ihre schädigenden Auswirkungen auf biologische Moleküle sind seit langem bekannt. Eine Reihe von Literaturberichten sieht einen Zusammenhang zwischen übermäßigem oxidativen Stress oder einer unzureichenden antioxidativen Verteidigung und Krebs, Atherosklerose und chronischen bzw. altersbedingten Erkrankungen. Mehrere Studien haben belegt, dass die Aktivierung des Renin-Angiotensin-Aldosteron-Systems zur Bildung von ROS führen kann. Epidemiologische Studien haben gezeigt, dass Nierenkarzinom-Inzidenzen und -Mortalitäten bei Hypertonikern erhöht sind. Vor kurzem konnte unsere Gruppe zeigen, dass die Perfusion von isolierten Maäusen-Nieren und dieoder Behandlung mehrerer Zelllinien mit Angiotensin II (Ang II) zur Bildung von DNA-Schäden und oxidativen Basenmodifikationen führt. Ziel der vorliegenden Arbeit war es, die Signalwege der Genotoxizität von Ang II zu bestimmen. Wir bestätigten dDie Genotoxiziät von Ang II in zwei Nieren-Zelllinien humaner Herkunft konnte bestätigt werden. Wir zeigten, dass Ang II-Behandlung zur Produktion von Superoxid-Anionen führt, die durch das membrangängige Superoxid-Dismutase-Mimetikum TEMPOL verhindert werden kann. Eines der Enzyme, das in den Zellen nach Ang II-Behandlung aktiviert wird und ROS produzieren kann, ist die NADPH-Oxidase. Die mittels RT-PCR gemessene Hochregulierung von p47 beweist die Aktivierung der NADPH-Oxidase nach Ang II-Behandlung. Auch die Phosphorylierung von p47 nach Ang II-Behandlung wurde gesteigert. Mittels zweier Inhibitoren zeigten wir, dass NADPH-Oxidase-Hemmung DNA-Schäden durch Ang II-Behandlung vollständig verhindert. Wir versuchten, die Rolle der Nox2- und Nox4-Isoformen der NADPH-Oxidase-Untereinheiten bei der Genotoxizität von Ang II zu differenzieren. Hemmung mittels siRNA bestätigte nur eine Beteiligung der Nox4. Anschließend überprüften wir die Rolle der PKC als potentiellem Aktivator der NADPH-Oxidase. Wir zeigten, dass die PKC nach Ang II-Behandlung PKC phosphoryliert wird und durch die Hemmung der PKC Ang II-induzierten Schäden verhindert werdenird. Die Verwendung mehrerer Inhibitoren der verschiedenen Teile des Signalweges zeigte, dass die PKC-Aktivierung von der Reaktion der PLC mit Membranphospholipiden und der Produktion von IP3 und DAG abhängig ist. IP3 bindet an seinen Rezeptor am Endoplasmatischen Retikulum (ER)., dDie in der Folge auftretende Öffnung eines Kanals ermöglicht einen Calcium-Ausstrom in das Cytoplasma. Auf diese Weise sind sowohl ER-Calcium als auch extrazelluläres Calcium an der Ang II-induzierten genotoxische Wirkung beteiligt. PLC wird durch AT1R-Stimulation aktiviert. Wir konnten mit Hilfe des AT1R-Antagonisten Candesartan auch zeigen, dass die Genotoxizität von Ang II über AT1R-Signaltransduktion vermittelt wird. Zusammenfassend haben wir gezeigt, dass Ang II genomische Schäden in humanen Nieren-Zelllinien verursacht. Die Schäden sind mit der Produktion von ROS verbunden. Eine Reduktion der Ang II-induzierten DNA-Schäden wurde nach Hemmung vonder G-Proteinen, der PLC, PKC und NADPH-Oxidase und Beeinflussung intra- sowie extrazellulärer Calium-Signalgebung gezeigt. Dies führt zu folgendem vorläufigen Modell der Signaltransduktion der von Ang II-induzierten DNA-Schäden: Die Bindung von Ang II an den AT1-Rezeptor aktiviert die PLC durch Stimulationerung der G-Proteine und die PKC in Calcium-abhängiger Weise, dies wiederum aktiviert die NADPH-Oxidase. Die NADPH Oxidase unter Beteiligung ihrerseiner Nox4-Untereinheit erzeugt dann reaktive Sauerstoffspezies, die DNA-Schäden verursachen. Dopamingehalt und -stoffwechsel in peripheren Lymphozyten von Parkinson-Patienten werden durch L-Dopa-Gabe beeinflusst. Die Patienten, die eine hohe Dosis L-Dopa erhalten, zeigen einen signifikant höheren Gehalt an Dopamin in den Lymphozyten im Vergleich zu Patienten, die eine niedrige Dosis L-Dopa erhalten oder der gesunden Kontrollgruppe. Im Mittelpunkt vieler Prozesse bei der Entstehung von oxidativem Stress und oxidativer Schäden bei Parkinson-Patienten steht die Monoaminoxidase (MAO), die für die enzymatische Oxidation von Dopamin und in der Folge für die Entstehung von H2O2 verantwortlich ist. Wir untersuchten, ob die Oxidation von Dopamin genotoxische Wirkung in Lymphozyten von Parkinson-Patienten mit hochdosierter L-Dopa-Therapie induzieren kann. Danach überprüftenfragten wir, ob die Behandlung mit Dopamin in vitro DNA-Schäden induzieren kann und versuchten aufzuzeigen, durch welchen Mechanismus Dopamin seine genotoxische Wirkung entfaltet. Die Häufigkeit von Mikrokernen in peripheren Lymphozyten der Parkinson-Patienten war nicht erhöht im Vergleich zur gesunden Kontrollgruppe, allerdings zeigte die Mikrokernfrequenz eine positive Korrelation mit der täglichen L-Dopa-Dosis bei Patienten, die eine L-Dopa-Therapie zusammen mit einem Dopamin-Rezeptor-Agonisten erhielten. In vitro beobachteten wir bei niedrigen mikromolaren Konzentrationen eine Induktion des genomischen Schadens in Zelllinien, die aus verschiedenen Geweben stammten. Die genotoxische Wirkung von Dopamin wurde durch Zugabe der Antioxidantien TEMPOL und DMTU reduziert, wodurch die Beteiligung von ROS gezeigt werden konnte. Um festzustellen, ob die Oxidation von Dopamin durch MAO für die Genotoxizität relevant ist, hemmten wir MAO mit zwei Inhibitoren, trans-2-Phenylcyclopropylamin-Hydrochlorid (PCPA) und Ro 16-6491, die beide die Bildung von Mikrokernen in PC-12-Zellen reduzieren konnten. Wir untersuchten auch die Rolle des Dopamin-Transporters (DAT) und Dopamin-Typ-2-Rezeptor (D2R)-assoziierter Signalwege in der Genotoxizität von Dopamin. Die Inhibitoren des DAT, GBR-12909 und Nomifensin verhinderten die Dopamin-induzierte Genotoxizität. Diese Ergebnisse wurden durch Behandlung von MDCK- und MDCK-DAT- Zellen (die das humane DAT-Gen besitzen) mit Dopamin bestätigt. Nur MDCK-DAT-Zellen zeigten erhöhte chromosomale Schäden und Dopaminaufnahme. Obwohl die Stimulation mit dem D2R-Rezeptor-Agonisten Quinpirol in Abwesenheit von Dopamin keine Genotoxizität in PC-12-Zellen induzierte, reduzierten sowohl ein D2R-Antagonist, wie auch Inhibitoren des in der Signalkaskade involvierten G-Proteins, der Phosphoinositol-3-Kinase und der extrazellulären signalregulierten Kinasen die Aufnahme von Dopamin mittels DAT und die Dopamin-vermittelte Genotoxizität. Der D2R-Antagonist Sulpirid hemmte die Dopamin-induzierte Migration von DAT aus dem Cytosol zur Zellmembran. Insgesamt verursacht der Neurotransmitter Dopamin DNA-Schäden und oxidativen Stress in vitro. Es gibt Hinweise, dass eine hochdosierte L-Dopa-Therapie zu oxidativem Stress führt. In vitro führt Dopamin zu Genotoxizität durch den Transport in die Zellen und Oxidation durch MAO. Der Transport von Dopamin durch DAT spielt eine zentrale Rolle in diesem Prozess. Die D2R-Signalwege sind an der Genotoxizität von Dopamin durch Auswirkung auf die Aktivierung und Membranexpression von DAT und damit der Dopaminaufnahme beteiligt.
KW  - Angiotensin II
KW  - Mutagenität
KW  - DNS-Schädigung
KW  - DNA-Schaden
KW  - Genotoxizität
KW  - genotoxicity
KW  - DNA damage
Y1  - 2010
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-55634
ER  - 
TY  - JOUR
A1  - Marzocco, Stefania
A1  - Fazeli, Gholamreza
A1  - Di Micco, Lucia
A1  - Autore, Giuseppina
A1  - Adesso, Simona
A1  - Dal Piaz, Fabrizio
A1  - Heidland, August
A1  - Di Iorio, Biagio
T1  - Supplementation of short-chain fatty acid, sodium propionate, in patients on maintenance hemodialysis: beneficial effects on inflammatory parameters and gut-derived uremic toxins, a pilot study (PLAN Study)
JF  - Journal of Clinical Medicine
N2  - Background: In end-stage renal disease (ESRD), gut-derived uremic toxins play a crucial role in the systemic inflammation and oxidative stress promoting the excess morbidity and mortality. The biochemical derangement is in part a consequence of an insufficient generation of short-chain fatty acids (SCFA) due to the dysbiosis of the gut and an insufficient consumption of the fermentable complex carbohydrates. Aim of the study: The primary end-point was to evaluate the potential efficacy of SCFA (specifically, sodium propionate (SP)) for patients on maintenance hemodialysis (MHD) on systemic inflammation. Secondary end-points included potential attenuation of oxidative stress markers, insulin resistance and production of gut-derived uremic toxins indoxyl sulfate and p-cresol sulfate, as well as health status after SP supplementation. Study design: We performed a single-center non-randomized pilot study in 20 MHD patients. They received the food additive SP with a daily intake of 2 × 500 mg in the form of capsules for 12 weeks. Pre-dialysis blood samples were taken at the beginning, after six weeks and at the end of the administration period, as well as four weeks after withdrawal of the treatment. Results: The subjects revealed a significant decline of inflammatory parameters C-reactive protein (−46%), interleukin IL-2 (−27%) and IL-17 (−15%). The inflammatory parameters IL-6 and IFN-gamma showed a mild non-significant reduction and the anti-inflammatory cytokine IL-10 increased significantly (+71%). While the concentration of bacterial endotoxins and TNF-α remained unchanged, the gut-derived uremic toxins, indoxyl sulfate (−30%) and p-cresyl sulfate (−50%), revealed a significant decline. The SP supplementation reduced the parameters of oxidative stress malondialdehyde (−32%) and glutathione peroxidase activity (−28%). The serum insulin levels dropped by 30% and the HOMA-index by 32%. The reduction of inflammatory parameters was associated with a lowering of ferritin and a significant increase in transferrin saturation (TSAT). Four weeks after the end of the treatment phase, all improved parameters deteriorated again. Evaluation of the psycho-physical performance with the short form 36 (SF-36) questionnaire showed an enhancement in the self-reported physical functioning, general health, vitality and mental health. The SP supplementation was well tolerated and without important side effects. No patient had left the study due to intolerance to the medication. The SP supplementation in MHD patients reduced pro-inflammatory parameters and oxidative stress and improved insulin resistance and iron metabolism. Furthermore, SP effectively lowered the important gut-derived uremic toxins indoxyl and p-cresol sulfate. These improvements were associated with a better quality of life. Further controlled studies are required in a larger cohort to evaluate the clinical outcome.
KW  - propionic acid
KW  - chronic kidney disease
KW  - hemodialysis
KW  - gut microbiome
KW  - systemic micro-inflammation oxidative stress
KW  - indoxyl sulfate
KW  - p-cresyl sulfate
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197626
SN  - 2077-0383
VL  - 7
IS  - 10
ER  - 
TY  - JOUR
A1  - Fazeli, Gholamreza
A1  - Beer, Katharina B.
A1  - Geisenhof, Michaela
A1  - Tröger, Sarah
A1  - König, Julia
A1  - Müller-Reichert, Thomas
A1  - Wehman, Ann M.
T1  - Loss of the Major Phosphatidylserine or Phosphatidylethanolamine Flippases Differentially Affect Phagocytosis
JF  - Frontiers in Cell and Developmental Biology
N2  - The lipids phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEth) are normally asymmetrically localized to the cytosolic face of membrane bilayers, but can both be externalized during diverse biological processes, including cell division, cell fusion, and cell death. Externalized lipids in the plasma membrane are recognized by lipid-binding proteins to regulate the clearance of cell corpses and other cell debris. However, it is unclear whether PtdSer and PtdEth contribute in similar or distinct ways to these processes. We discovered that disruption of the lipid flippases that maintain PtdSer or PtdEth asymmetry in the plasma membrane have opposite effects on phagocytosis in Caenorhabditis elegans embryos. Constitutive PtdSer externalization caused by disruption of the major PtdSer flippase TAT-1 led to increased phagocytosis of cell debris, sometimes leading to two cells engulfing the same debris. In contrast, PtdEth externalization caused by depletion of the major PtdEth flippase TAT-5 or its activator PAD-1 disrupted phagocytosis. These data suggest that PtdSer and PtdEth externalization have opposite effects on phagocytosis. Furthermore, externalizing PtdEth is associated with increased extracellular vesicle release, and we present evidence that the extent of extracellular vesicle accumulation correlates with the extent of phagocytic defects. Thus, a general loss of lipid asymmetry can have opposing impacts through different lipid subtypes simultaneously exerting disparate effects.
KW  - phagocytosis
KW  - lipid asymmetry
KW  - flippase
KW  - phosphatidylserine
KW  - phosphatidylethanolamine
KW  - extracellular vesicle
Y1  - 2020
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-208771
SN  - 2296-634X
VL  - 8
ER  -