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.

Oxygen-charged HTK-F6H8 emulsion reduces ischemia-reperfusion injury in kidneys from brain-dead pigs.

BACKGROUND: Prolonged cold ischemia is frequently associated with a greater risk of delayed graft function and enhanced graft failure. We hypothesized that media, combining a high oxygen-dissolving capacity with specific qualities of organ preservation solutions, would be more efficient in reducing immediate ischemia-reperfusion injury from organs stored long term compared with standard preservation media. METHODS: Kidneys retrieved from brain-dead pigs were flushed using either cold histidine-tryptophan-ketoglutarate (HTK) or oxygen-precharged emulsion composed of 75% HTK and 25% perfluorohexyloctane. After 18 h of cold ischemia the kidneys were transplanted into allogeneic recipients and assessed for adenosine triphosphate content, morphology, and expression of genes related to hypoxia, environmental stress, inflammation, and apoptosis. RESULTS: Compared with HTK-flushed kidneys, organs preserved using oxygen-precharged HTK-perfluorohexyloctane emulsion had increased elevated adenosine triphosphate content and a significantly lower gene expression of hypoxia inducible factor-1α, vascular endothelial growth factor, interleukin-1α, tumor necrosis factor-α, interferon-α, JNK-1, p38, cytochrome-c, Bax, caspase-8, and caspase-3 at all time points assessed. In contrast, the mRNA expression of Bcl-2 was significantly increased. CONCLUSIONS: The present study has demonstrated that in brain-dead pigs the perfusion of kidneys with oxygen-precharged HTK-perfluorohexyloctane emulsion results in significantly reduced inflammation, hypoxic injury, and apoptosis and cellular integrity and energy content are well maintained. Histologic examination revealed less tubular, vascular, and glomerular changes in the emulsion-perfused tissue compared with the HTK-perfused counterparts. The concept of perfusing organs with oxygen-precharged emulsion based on organ preservation media represents an efficient alternative for improved organ preservation.

Inhibition of VCAM-1 expression in endothelial cells by CORM-3: the role of the ubiquitin-proteasome system, p38, and mitochondrial respiration.

Carbon monoxide (CO) abrogates TNF-α-mediated inflammatory responses in endothelial cells, yet the underlying mechanism thereof is still elusive. We have previously shown that the anti-inflammatory effect of CO-releasing molecule-3 (CORM-3) is not completely mediated via deactivation of the NF-κB pathway. In this study, we sought to explore other potential mechanisms by which CORM-3 downregulates VCAM-1 expression on TNF-α-stimulated HUVECs. By genome-wide gene expression profiling and pathway analysis we studied the relevance of particular pathways for the anti-inflammatory effect of CORM-3. In CORM-3-stimulated HUVECs significant changes in expression were found for genes implicated in the proteasome and porphyrin pathways. Although proteasome activities were increased by CORM-3, proteasome inhibitors did not abolish the effect of CORM-3. Likewise, heme oxygenase-1 inhibitors did not abrogate the ability of CORM-3 to downregulate VCAM-1 expression. Interestingly, CORM-3 inhibited MAPK p38, and the p38 inhibitor SB203580 downregulated VCAM-1 expression. However, downregulation of VCAM-1 by CORM-3 occurred only at concentrations that partly inhibit ATP production and sodium azide and oligomycin paralleled the effect of CORM-3 in this regard. Our results indicate that CORM-3-induced downregulation of VCAM-1 is mediated via p38 inhibition and mitochondrial respiration, whereas the ubiquitin-proteasome system seems not to be involved.

Donor desmopressin is associated with superior graft survival after kidney transplantation.

BACKGROUND: A recent randomized trial showed that pretreatment of the brain-dead donor with low-dose dopamine improves immediate kidney graft function, by limiting injury from cold storage (ClinicalTrials.gov Identifier: NCT00115115). This study determines whether donor exposure to desmopressin (1-deamino-8-d-arginine-vasopressin [DDAVP]) before organ retrieval affects renal transplant outcome. METHODS: This retrospective multicenter cohort study, nested in the database of the dopamine trial, includes 264 deceased heart-beating donors with confirmed brain death and corresponding 487 renal allograft recipients transplanted at 60 European centers between March 2004 and August 2007. We assessed differences in delayed graft function, biopsy-proven acute rejections, and 2-year kidney graft survival in recipients of a DDAVP-exposed versus unexposed graft. RESULTS: DDAVP was associated with improved graft survival (85.4% vs. 73.6%, P=0.003). This survival benefit persisted after censoring for death with functioning graft (91.1% vs. 82.0%, P=0.01) and after adjustment for confounders including covariate adjustment from propensity scoring (hazard ratio 0.40, 95% confidence interval [CI] 0.21-0.77; P=0.006). Delayed graft function (odds ratio 0.97, 95% CI 0.57-1.65; P=0.92) and biopsy-proven acute rejections (odds ratio 1.32, 95% CI 0.70-2.49; P=0.40) were unaffected. The survival effect was enhanced after a shorter cold ischemic time less than 14 hr (91.3% vs. 77.8%, P=0.008) and after dopamine pretreatment (92.7% vs. 78.6%, P=0.006). By contrast, prolonged cold ischemic time more than or equal to 14 hr (91.2% vs. 86.5%, P=0.39) and assignment to the nondopamine group (89.7% vs. 84.8%, P=0.37) abrogated the survival advantage. CONCLUSIONS: Donor DDAVP seems to improve renal allograft survival. Combined use of donor DDAVP and low-dose dopamine should receive further evaluation.

Effects of donor pre-treatment with dopamine on survival after heart transplantation: a cohort study of heart transplant recipients nested in a randomized controlled multicenter trial.

OBJECTIVES: We determined the outcome of cardiac allografts from multiorgan donors enrolled in a randomized trial of donor pre-treatment with dopamine. BACKGROUND: Treatment of the brain-dead donor with low-dose dopamine improves immediate graft function after kidney transplantation. METHODS: A cohort study of 93 heart transplants from 21 European centers was undertaken between March 2004 and August 2007. We assessed post-transplant left ventricular function (LVF), requirement of a left ventricular assist device (LVAD) or biventricular assist device (BVAD), need for hemofiltration, acute rejection, and survival of recipients of a dopamine-treated versus untreated graft. RESULTS: Donor dopamine was associated with improved survival 3 years after transplantation (87.0% vs. 67.8%, p = 0.03). Fewer recipients of a pre-treated graft required hemofiltration after transplant (21.7% vs. 40.4%, p = 0.05). Impaired LVF (15.2% vs. 21.3%, p = 0.59), requirement of a LVAD (4.4% vs. 10.6%, p = 0.44), and biopsy-proven acute rejection (19.6% vs. 14.9%, p = 0.59) were not statistically different between groups. Post-transplant impaired LVF (hazard ratio [HR]: 4.95; 95% confidence interval [CI]: 2.08 to 11.79; p < 0.001), requirement of LVAD (HR: 6.65; 95% CI: 2.40 to 18.45; p < 0.001), and hemofiltration (HR: 2.83; 95% CI: 1.20 to 6.69; p = 0.02) were predictive of death. The survival benefit remained (HR: 0.33; 95% CI: 0.12 to 0.89; p = 0.03) after adjustment for various risks affecting mortality, including pre-transplant LVAD/BVAD, inotropic support, and impaired kidney function. CONCLUSIONS: Treatment of brain-dead donors with dopamine of 4 µg/kg/min will not harm cardiac allografts but appears to improve the clinical course of the heart allograft recipient. (Prospective Randomized Trial to Evaluate the Efficacy of Donor Preconditioning With Dopamine on Initial Graft Function After Kidney Transplantation; NCT00115115).

UW is superior compared with HTK after prolonged preservation of renal grafts.

BACKGROUND: In recent clinical studies, the efficacy of histidine-tryptophan-ketoglutarate (HTK) in kidney transplantation was questioned. This study compares the efficacy of University of Wisconsin (UW) and HTK solutions on transplantation outcome. MATERIALS AND METHODS: Rat kidneys were preserved for different periods of cold ischemia (CIT). Heat capacity of the solutions, temperature of the grafts, renal function (RF), and histology were assessed before and after transplantation, respectively. RESULTS: After prolonged CIT, recipient survival was superior in the UW - (100%) compared with the HTK group (10%). In the latter, severe tubular necrosis, DNA damage, and renal inflammation were observed, reflected by an increased KIM-1, IL6, and P-selectin expression. CIT correlated negatively with RF in both groups. RF recovered significantly faster in the UW group. LDH-release and ATP depletion after cold storage of tubular cells were lower in UW than in HTK. Heat capacity was significantly higher for UW than for HTK. Accordingly, renal temperature was lower. CONCLUSIONS: Prolonged preservation in UW solution results in a better renal function and less tissue damage compared with HTK, possibly due to improved cooling and better cell viability of the graft. The use of HTK for renal allografts should therefore be reconsidered, particularly when CIT is expected to be long.

Effects of N-acetylcysteine on renal hemodynamics in contrast media-induced nephropathy.

BACKGROUND: N-acetylcysteine (NAC) has been proposed to prevent radiocontrast nephropathy in high-risk patients. METHODS: The effect of single-dose and prolonged administration of NAC before application of either the ionic, high-osmolar radiocontrast agent diatrizoate sodium (DTZ) or the nonionic, low-osmolar radiocontrast agent iohexol (IOH) in a rat model combining uninephrectomy, salt depletion, and administration of indomethacin was explored. Arterial blood pressure and total, cortical, and medullary blood flow were continuously recorded in anesthetized Sprague-Dawley rats. RESULTS: NAC had no effect on renal hemodynamics in control rats. Both DTZ and IOH induced biphasic changes in renal blood flow and cortical renal blood flux and persistently reduced medullary blood flux. Neither single-dose nor prolonged administration of NAC prevented the hemodynamic changes following administration of DTZ or IOH, respectively. Acute prophylactic administration of NAC prevented increased urinary ET excretion after injection of IOH and, to a smaller degree, of DTZ. Both an ionic, high-osmolar (DTZ) and a nonionic, low-osmolar (IOH) radiocontrast agent induce marked changes in renal hemodynamics in salt-depleted rats treated with indomethacin. CONCLUSIONS: Renal perfusion is not affected by NAC application in a model of experimental contrast nephropathy in rats. Other effects of NAC might thus account for the presumed renoprotective properties.

Donor dopamine treatment in brain dead rats is associated with an improvement in renal function early after transplantation and a reduction in renal inflammation.

Brain death (BD) is associated with tissue inflammation. As dopamine treatment of BD donor rats reduces renal monocyte infiltration, we tested if this treatment affects renal function and inflammation in recipients. BD was induced in F344 rats and was maintained for 6 h in all experiments. Dopamine was given for 6 (DA6) or 3 h (DA3) from the onset of BD. Ventilated non-BD (NBD) and BD animals served as controls. Kidneys were transplanted into bilaterally nephrectomized Lewis recipients. Serum creatinine (s-crea) was measured and leukocyte infiltration was assessed 10 days after transplantation. One day after transplantation, s-crea was significantly reduced in recipients who received a renal allograft from dopamine treated BD or from NBD rats compared to BD vehicle (P < 0.05). Ten days after transplantation, the number of infiltrating monocytes was significantly lower in grafts obtained from dopamine treated and from NBD rats (P < 0.05). A reduced infiltration in these grafts was confirmed by Banff 97 classification. Cytokine-induced neutrophil-chemoattractant 1 and interleukin (IL)-6 mRNA expression were reduced in DA rats compared to BD controls. No difference for macrophage chemoattractant protein 1 and IL-10 were found. These findings may explain the salutary effect of donor dopamine treatment in renal transplantation.

Donor dopamine pretreatment inhibits tubulitis in renal allografts subjected to prolonged cold preservation.

BACKGROUND: In the present study, we used the Brown-Norway (BN) to Lewis model as a model for acute rejection, to test the hypothesis that dopamine (DA) treatment of BN donors significantly reduces the inflammatory response after renal transplantation. METHODS: BN and Lewis rats (isograft controls) were treated for 24 hr with DA (5 microg/kg/min) or NaCl (0.9%), respectively. After 24 hr of cold storage in University of Wisconsin (UW) solution, renal allografts were orthotopically transplanted into Lewis recipients. All recipients received immunosuppression until they were sacrificed. Allografts were harvested one, three, five, and 10 days after transplantation and analyzed by light microscopy, immunohistochemistry (CD3, major histocompatibility complex [MHC] class II, ED1, P-selectin and intercellular adhesion molecule [ICAM]-1) and by RNase protection assay for cytokine mRNA. RESULTS: Ten days after transplantation Banff tubulitis scores were significantly lower in DA-treated than in NaCl-treated allografts. No significant differences were found in Banff interstitial infiltration scores. The numbers of MHC class II+ and CD3+ cells were significantly decreased in DA-treated animals as assessed by immunohistochemistry. No differences were found in the number of ED1+, P-selectin+, and ICAM-1+ cells. The expression of Ltalpha, tumor necrosis factor, interleukin-1beta, and interleukin-2 mRNA was significantly reduced in DA-treated animals. CONCLUSION: Our data indicate that donor DA treatment significantly inhibits tubulitis in renal allografts subjected to prolonged cold preservation. A reduced number of infiltrating MHC class II+ and CD3+ cells together with decreased cytokine expression could diminish renal scarring, reduce allograft immunogenicity, and hence improve transplantation outcome.