Martin Alexander Schick

Rosiglitazone Affects Nitric Oxide Synthases and Improves Renal Outcome in a Rat Model of Severe Ischemia/Reperfusion Injury

Background. Nitric oxide (NO)-signal transduction plays an important role in renal ischemia/reperfusion (I/R) injury. NO produced by endothelial NO-synthase (eNOS) has protective functions whereas NO from inducible NO-synthase (iNOS) induces impairment. Rosiglitazone (RGZ), a peroxisome proliferator-activated receptor (PPAR)-γ agonist exerted beneficial effects after renal I/R injury, so we investigated whether this might be causally linked with NOS imbalance. Methods. RGZ (5 mg/kg) was administered i.p. to SD-rats (f) subjected to bilateral renal ischemia (60 min). Following 24 h of reperfusion, inulin- and PAH-clearance as well as PAH-net secretion were determined. Morphological alterations were graded by histopathological scoring. Plasma NOx-production was measured. eNOS and iNOS expression was analyzed by qPCR. Cleaved caspase 3 (CC3) was determined as an apoptosis indicator and ED1 as a marker of macrophage infiltration in renal tissue. Results. RGZ improves renal function after renal I/R injury (PAH-/inulin-clearance, PAH-net secretion) and reduces histomorphological injury. Additionally, RGZ reduces NOx plasma levels, ED-1 positive cell infiltration and CC3 expression. iNOS-mRNA is reduced whereas eNOS-mRNA is increased by RGZ. Conclusion. RGZ has protective properties after severe renal I/R injury. Alterations of the NO pathway regarding eNOS and iNOS could be an explanation of the underlying mechanism of RGZ protection in renal I/R injury.

Phosphodiesterase-4 inhibition as a therapeutic approach to treat capillary leakage in systemic inflammation

•  A specific therapy to treat capillary leakage in systemic inflammation and sepsis is not available at present. •  Recent studies demonstrated that reduced cAMP levels in endothelial cells contribute to inflammation‐induced breakdown of the endothelial barrier. •  The present study demonstrates that systemically applied phosphodiesterase‐4 inhibitors to increase endothelial cAMP are effective to prevent and to treat capillary leakage followed by improved microcirculation in a rodent model of systemic inflammation. •  These data suggest a highly clinically relevant and applicable approach to stabilize capillary leakage in sepsis and systemic inflammation.

Soluble VE-cadherin is involved in endothelial barrier breakdown in systemic inflammation and sepsis

AIMS Microvascular endothelial barrier breakdown in sepsis precedes organ failure and death in patients. We tested the hypothesis that the formation of endothelium-derived soluble vascular endothelial (VE)-cadherin fragments (sVE-cadherin) is involved in inflammation-induced endothelial barrier disruption. METHODS AND RESULTS Incubation of human dermal microvascular endothelial cells (HDMEC) with tumour necrosis factor-α (TNF-α) and bacterial lipopolysaccharide (LPS) led to endothelial barrier disruption which correlated with significantly increased sVE-cadherin at a size of ∼90 kDa in cell culture supernatants. Inhibition of the VE-cadherin-cleaving disintegrin and metalloproteinase ADAM10 using GI254023X attenuated inflammation-induced formation of sVE-cadherin and endothelial barrier disruption, suggesting ADAM10-mediated shedding as a mechanism underlying sVE-cadherin release. Formation of VE-cadherin fragments at 90 and 110 kDa was observed when recombinant VE-cadherin (rVE-cadherin) was digested with recombinant ADAM10. Mass spectrometry of the VE-cadherin fragments showed that they originated from cleavage of the extracelluar domain and thereby several cleavage sites of ADAM10 were identified. Atomic force microscopy measurements demonstrated that cell culture supernatants containing sVE-cadherin and application of rVE-cadherin blocked VE-cadherin binding. Accordingly rVE-cadherin dose-dependently led to loss of endothelial barrier functions in HDMEC monolayers. Finally, in patients suffering from severe sepsis or septic shock with clinical signs of a microvascular leackage, serum levels of sVE-cadherin were significantly increased. CONCLUSION Taken together, formation of sVE-cadherin is associated and contributes to inflammation-induced breakdown of endothelial barrier functions by inhibition of VE-cadherin binding. The underlying mechanism of VE-cadherin cleavage involves ADAM10 and appears to be of clinical relevance since sVE-cadherin was augmented in patients with severe sepsis.

The effects of colloid solutions on renal proximal tubular cells in vitro.

Renal failure is a common complication of critically ill patients. Colloids such as hydroxyethyl starch (HES), gelatin, or albumin are regularly used for intravascular volume resuscitation, but there are increasing reports about the nephrotoxic side effects of synthetic colloids in septic patients. Therefore, we investigated the influence of colloids (HES130/0.4 (Voluven®), gelatin (Gelafundin®), human albumin, and the crystalloid Sterofundin® ISO on cell viability of human proximal tubular (HK-2) cells. HK-2 cells were incubated with colloids (0.1%–4%) and with equivalent volumes of the crystalloid solution Sterofundin ISO. After 21 hours, cell viability of HK-2 cells was measured by EZ4U assay (dye XTT). Application of HES130/0.4 decreased cell viability significantly in a concentration-dependent manner (86.80% ± 10.79% by 0.5% HES down to 24.02% ± 4.27% by 4% HES). Human albumin (>1.25%) as well as gelatin (>1%) also showed deleterious effects on HK-2 cells. Interestingly, in lower concentrations, human albumin and the crystalloid solution Sterofundin ISO were cytoprotective in comparison with the NaCl control. In conclusion, synthetic and natural colloids showed a harmful impact on HK-2 cells in higher concentrations without any prior proinflammatory stimulus. HES130/0.4 exhibited the most distinctive harmful impact, whereas the application of crystalloid Sterofundin ISO revealed cytoprotective effects.

Carbon monoxide has antioxidative properties in the liver involving p38 MAP kinase pathway in a murine model of systemic inflammation.

Please cite this paper as: Brugger, Schick, Brock, Baumann, Muellenbach, Roewer and Wunder (2010). Carbon Monoxide has Antioxidative Properties in the Liver Involving p38 MAP Kinase Pathway in a Murine Model of Systemic Inflammation. Microcirculation17(7), 504–513.

Effects of crystalloids and colloids on liver and intestine microcirculation and function in cecal ligation and puncture induced septic rodents

BackgroundSeptic acute liver and intestinal failure is associated with a high mortality. We therefore investigated the influence of volume resuscitation with different crystalloid or colloid solutions on liver and intestine injury and microcirculation in septic rodents.MethodsSepsis was induced by cecal ligation and puncture (CLP) in 77 male rats. Animals were treated with different crystalloids (NaCl 0.9% (NaCl), Ringer’s acetate (RA)) or colloids (Gelafundin 4% (Gel), 6% HES 130/0.4 (HES)). After 24 h animals were re-anesthetized and intestinal (n = 6/group) and liver microcirculation (n = 6/group) were obtained using intravital microscopy, as well as macrohemodynamic parameters were measured. Blood assays and organs were harvested to determine organ function and injury.ResultsHES improved liver microcirculation, cardiac index and DO2-I, but significantly increased IL-1β, IL-6 and TNF-α levels and resulted in a mortality rate of 33%. Gel infused animals revealed significant reduction of liver and intestine microcirculation with severe side effects on coagulation (significantly increased PTT and INR, decreased haemoglobin and platelet count). Furthermore Gel showed severe hypoglycemia, acidosis and significantly increased ALT and IL-6 with a lethality of 29%. RA exhibited no derangements in liver microcirculation when compared to sham and HES. RA showed no intestinal microcirculation disturbance compared to sham, but significantly improved the number of intestinal capillaries with flow compared to HES. All RA treated animals survided and showed no severe side effects on coagulation, liver, macrohemodynamic or metabolic state.ConclusionsGelatine 4% revealed devastated hepatic and intestinal microcirculation and severe side effects in CLP induced septic rats, whereas the balanced crystalloid solution showed stabilization of macro- and microhemodynamics with improved survival. HES improved liver microcirculation, but exhibited significantly increased pro-inflammatory cytokine levels. Crystalloid infusion revealed best results in mortality and microcirculation, when compared with colloid infusion.

Phosphodiesterase 4 inhibition dose dependently stabilizes microvascular barrier functions and microcirculation in a rodent model of polymicrobial sepsis.

ABSTRACT Background: Breakdown of microvascular endothelial barrier functions contributes to disturbed microcirculation, organ failure, and death in sepsis. Increased endothelial cAMP levels by systemic application of phosphodiesterase 4 inhibitors (PD-4-I) have previously been demonstrated to protect microvascular barrier properties in a model of systemic inflammation (systemic inflammatory response syndrome) suggesting a novel therapeutic option to overcome this problem. However, in a clinically relevant model of polymicrobial sepsis long-term effects, immunomodulatory effects and effectivity of PD-4-I to stabilize microvascular barrier functions and microcirculation remained unexplored. Methods: We induced polymicrobial sepsis using the colon ascendens stent peritonitis (CASP) model in which we performed macrohemodynamic and microhemodynamic monitoring with and without systemic intravenous application of different doses of PD-4-I rolipram in Sprague-Dawley rats over 26 h. Results: All animals with CASP showed clinical and laboratory signs of sepsis and peritonitis. Whereas macrohemodynamic adverse effects were not evident, application of PD-4-I led to stabilization of endothelial barrier properties as revealed by reduced extravasation of fluorescein isothiocyanate–albumin. However, only low-dose application of 1 mg/kg body weight per hour of PD-4-I improved microcirculatory flow in the CASP model, whereas high-dose therapy of 3 mg/kg BW per hour PDI-4-I had adverse effects. Accordingly, sepsis-induced acute kidney injury and lung edema were prevented by PD-4-I treatment. Furthermore, PD-4-I showed immunomodulatory effects as revealed by decreased interleukin 1&agr; (IL-1&agr;), IL-1&bgr;, IL-12, and tumor necrosis factor &agr; levels following PD-4-I treatment, which appeared not to correlate with barrier-stabilizing effects of rolipram. Conclusions: These data provide further evidence that systemic application of PD-4-I could be suitable for therapeutic microvascular barrier stabilization and improvement of microcirculatory flow in sepsis.

Activation of myenteric glia during acute inflammation in vitro and in vivo

Background Enteric glial cells (EGCs) are the main constituent of the enteric nervous system and share similarities with astrocytes from the central nervous system including their reactivity to an inflammatory microenvironment. Previous studies on EGC pathophysiology have specifically focused on mucosal glia activation and its contribution to mucosal inflammatory processes observed in the gut of inflammatory bowel disease (IBD) patients. In contrast knowledge is scarce on intestinal inflammation not locally restricted to the mucosa but systemically affecting the intestine and its effect on the overall EGC network. Methods and Results In this study, we analyzed the biological effects of a systemic LPS-induced hyperinflammatory insult on overall EGCs in a rat model in vivo, mimicking the clinical situation of systemic inflammation response syndrome (SIRS). Tissues from small and large intestine were removed 4 hours after systemic LPS-injection and analyzed on transcript and protein level. Laser capture microdissection was performed to study plexus-specific gene expression alterations. Upon systemic LPS-injection in vivo we observed a rapid and dramatic activation of Glial Fibrillary Acidic Protein (GFAP)-expressing glia on mRNA level, locally restricted to the myenteric plexus. To study the specific role of the GFAP subpopulation, we established flow cytometry-purified primary glial cell cultures from GFAP promotor-driven EGFP reporter mice. After LPS stimulation, we analyzed cytokine secretion and global gene expression profiles, which were finally implemented in a bioinformatic comparative transcriptome analysis. Enriched GFAP+ glial cells cultured as gliospheres secreted increased levels of prominent inflammatory cytokines upon LPS stimulation. Additionally, a shift in myenteric glial gene expression profile was induced that predominantly affected genes associated with immune response. Conclusion and Significance Our findings identify the myenteric GFAP-expressing glial subpopulation as particularly susceptible and responsive to acute systemic inflammation of the gut wall and complement knowledge on glial involvement in mucosal inflammation of the intestine.

Balanced Hydroxyethylstarch (HES 130/0.4) Impairs Kidney Function In-Vivo without Inflammation

Volume therapy is a standard procedure in daily perioperative care, and there is an ongoing discussion about the benefits of colloid resuscitation with hydroxyethylstarch (HES). In sepsis HES should be avoided due to a higher risk for acute kidney injury (AKI). Results of the usage of HES in patients without sepsis are controversial. Therefore we conducted an animal study to evaluate the impact of 6% HES 130/0.4 on kidney integrity with sepsis or under healthy conditions Sepsis was induced by standardized Colon Ascendens Stent Peritonitis (sCASP). sCASP-group as well as control group (C) remained untreated for 24 h. After 18 h sCASP+HES group (sCASP+VOL) and control+HES (C+VOL) received 50 ml/KG balanced 6% HES (VOL) 130/0.4 over 6h. After 24h kidney function was measured via Inulin- and PAH-Clearance in re-anesthetized rats, and serum urea, creatinine (crea), cystatin C and Neutrophil gelatinase-associated lipocalin (NGAL) as well as histopathology were analysed. In vitro human proximal tubule cells (PTC) were cultured +/- lipopolysaccharid (LPS) and with 0.1–4.0% VOL. Cell viability was measured with XTT-, cell toxicity with LDH-test. sCASP induced severe septic AKI demonstrated divergent results regarding renal function by clearance or creatinine measure focusing on VOL. Soleley HES (C+VOL) deteriorated renal function without sCASP. Histopathology revealed significantly derangements in all HES groups compared to control. In vitro LPS did not worsen the HES induced reduction of cell viability in PTC cells. For the first time, we demonstrated, that application of 50 ml/KG 6% HES 130/0.4 over 6 hours induced AKI without inflammation in vivo. Severity of sCASP induced septic AKI might be no longer susceptible to the way of volume expansion.

Molecular size and origin do not influence the harmful side effects of hydroxyethyl starch on human proximal tubule cells (HK-2) in vitro.

BACKGROUND:Recently, clinical trials revealed renal impairment induced by hydroxyethyl starch (HES) in septic patients. In prior studies, we managed to demonstrate that HES accumulated in renal proximal tubule cells (PTCs). The related pathomechanism has not yet been discovered. To validate our hypothesis that the HES molecule itself is harmful, regardless of its molecule size or origin, we conducted a comprehensive study to elucidate the influences of different HES preparations on PTC viability in vitro. METHODS:Cell viability of human PTC was measured with a cytotoxicity assay, quantifying the reduction of tetrazolium salt to colored formazan. Experiments were performed by assessing the influence of different carrier solutions of HES (balanced, nonbalanced, culture medium), different average molecular weights (70, 130, 200 kDa), different origins (potato or corn derived), and various durations of incubation (2–21 hours). Furthermore, HES 130/0.4 was fractionated by ultrafiltration, and the impact on cell viability of average single-size fractions with <3, 3 to 10, 10 to 30, 30 to 50, 50 to 100, and >100 kDa was investigated. We also tested the possible synergistic effects of inflammation induced by tumor necrosis factor-&agr;. RESULTS:All tested HES solutions, regardless of origin or carrier matrix, decreased cell viability in an equivalent, dose-dependent manner. Coincubation with tumor necrosis factor-&agr; did not reduce HES-induced reduction of cell viability. Minor differences were detected comparing 70, 130, and 200 kDa preparations. Analysis of fractionated HES revealed that each fraction decreased cell viability. Even small HES molecules (10–30 kDa) were significantly deleterious. CONCLUSIONS:For the first time, we were able to show that only the total mass of HES molecules applied is responsible for the harmful impact on renal PTC in vitro. Neither molecular size nor their origin showed any relevance.