Local renal complement C3 induction by donor brain death is associated with reduced renal allograft function after transplantation.

BACKGROUND: Kidneys derived from brain-dead donors have inferior outcomes after transplantation compared to kidneys from living donors. Strikingly, early and profound serum levels of IL-6 in brain-dead donors are observed. IL-6 is the main regulator of the acute phase response (APR). The aim of this translational study was to investigate the expression of renal acute phase proteins (APPs) following brain death (BD) and to assess the association with renal allograft outcome after transplantation. METHODS: BD was induced in rats by inflating a subdurally placed balloon catheter. Kidney biopsies were obtained from human living and brain-dead donors at donation, after cold preservation and reperfusion. In vitro, renal proximal tubular epithelial cells (HK-2 cells) were stimulated with IL-6. RESULTS: Both in human and rat brain-dead donors, C3 and FBG expression was enhanced at donation compared to living donors and sham-operated animals. In human donors, no additional expression was found after cold ischaemia or reperfusion. C3 expression after reperfusion was independently associated with decreased short-term function after transplantation in grafts from brain-dead donors. In cultured HK-2 cells, C3 production was induced in the presence of IL-6. CONCLUSIONS: In conclusion, BD induces renal C3 and FBG expression. Moreover, C3 expression is associated with a worse allograft function early after transplantation. Therefore, targeting renal APPs in brain-dead donors, especially complement C3, may improve transplant outcome.

Does hepatitis C increase the accumulation of advanced glycation end products in haemodialysis patients?

BACKGROUND: Hepatitis C may cause increased levels of oxidative stress that contribute to accumulation of advanced glycation end products (AGEs), which increase the risk of cardiovascular disease (CVD). The aim of this study was to determine the influence of hepatitis C on AGE accumulation in haemodialysis patients. METHODS: AGE accumulation was measured by means of skin autofluorescence (AF) in 92 haemodialysis (HD) patients and 93 age-matched healthy controls. In the HD patients, CVD-related biochemical variables were also measured. The HD patients were tested for hepatitis C virus (HCV) antibodies and allocated to a HCV+ or HCV- group. RESULTS: Skin AF of the healthy subjects was lower than skin AF in the HD patients (3.13 +/- 0.95 vs 2.2 +/- 0.47; P < 0.001). We calculated the average increase of skin AF in the healthy subjects to be 0.017 arbitrary units per year, being 14 times lower than in HD patients with CVD only and 20 times lower than in HD patients suffering from combined CVD and diabetes mellitus (DM). Multivariate regression analysis showed that AGE accumulation in HD patients can be described by the independent effects of age, DM, CVD and HD vintage. Although inter-cellular adhesion molecule 1 and liver enzymes were elevated in HCV+ HD patients, levels of oxidative stress markers and skin AF were not significantly different between HCV+ and HCV- HD patients. CONCLUSIONS: AGE accumulation was higher in the HD patients than in the healthy controls. AGE accumulation did not differ in HCV+ and HCV- HD patients. This might be due to the fact that hepatitis C did not cause oxidative stress in our HD population. Independent markers of AGE accumulation were age, HD vintage, DM and CVD, but not hepatitis C.

Morphine-6beta-glucuronide rapidly increases pain sensitivity independently of opioid receptor activity in mice and humans.

BACKGROUND: Previous data indicate that morphine-6beta-glucuronide (M6G), a morphine metabolite with analgesic properties, can paradoxically increase pain sensitivity in mice and humans. The authors tested mice and humans for M6G hyperalgesia and assessed the contribution of N-methyl-D-aspartate receptor activity in mice. METHODS: Nociception after acute injection (10 mg/kg) and chronic infusion (1.6 mg/kg per 24 h) of M6G or saline was assayed using the tail-withdrawal test in CD-1 mice implanted with pellets containing the opioid antagonist naltrexone or placebo and in knockout mice lacking mu-, kappa-, and delta-opioid receptors and their B6129F(1) controls. In volunteers, responses to heat pain were tested after a M6G (0.4 mg/kg) injection in the presence of a continuous high naloxone (0.04-mg/kg bolus followed by 0.04 mg/kg per hour) or saline background infusion. RESULTS: Acute M6G injection evoked analgesia in CD-1 mice implanted with placebo pellets and B6129F(1) control mice, whereas it caused hyperalgesia in CD-1 mice treated concurrently with naltrexone and in knockout mice. Continuous M6G infusion produced hyperalgesia within 24 h, lasting for a minimum of 6 days, in both placebo- and naltrexone-pelleted mice. The N-methyl-D-aspartate receptor antagonist MK-801 (0.05 mg/kg) blocked and reversed hyperalgesia after the acute injection and continuous infusion of M6G, respectively. In humans, M6G increased heat pain sensitivity for at least 6 h independently of simultaneous naloxone infusion. CONCLUSIONS: These data indicate that M6G causes hyperalgesia independent of previous or concurrent opioid receptor activity or analgesia. In mice, a causal role for the N-methyl-D-aspartate receptor is also indicated.

Early events in kidney donation: progression of endothelial activation, oxidative stress and tubular injury after brain death.

Cerebral injury leading to brain death (BD) causes major physiologic derangements in potential organ donors, which may result in vascular-endothelial activation and affect posttransplant graft function. We investigated the kinetic of pro-coagulatory and pro-inflammatory endothelial activation and the subsequent oxidative stress and renal tubular injury, early after BD declaration. BD was induced by slowly inflating a balloon-catheter inserted in the extradural space over a period of 30 min. Rats (n = 30) were sacrificed 0.5, 1, 2 or 4 h after BD-induction and compared with sham-controls. This study demonstrates immediate pro-coagulatory and pro-inflammatory activation of vascular endothelium after BD in kidney donor rats, proportional with the duration of BD. E- and P-Selectins, Aalpha/Bbeta-fibrinogen mRNA were abruptly and progressively up-regulated from 0.5 h BD onwards; P-Selectin membrane protein expression was increased; fibrinogen was primarily visualized in the peritubular capillaries. Plasma von Willebrand factor was significantly higher after 2 h and 4 h BD. Urine heart-fatty-acid-binding-protein and N-acetyl-glucosaminidase, used as new specific and sensitive markers of proximal and distal tubular damage, were found significantly increased after 0.5 h, with a maximum at 4 h. Unexpectedly, oxidative stress was detectable only late, after the installation of tubular injury, suggesting only a secondary role for hypoxia in triggering these injuries.

Attenuated renal and intestinal injury after use of a mini-cardiopulmonary bypass system.

BACKGROUND: Transient, subclinical myocardial, renal, intestinal, and hepatic tissue injury and impaired homeostasis is detectable even in low-risk patients undergoing conventional cardiopulmonary bypass (CPB). Small extracorporeal closed circuits with low priming volumes and optimized perfusion have been developed to reduce deleterious effects of CPB. METHODS: A prospective, randomized trial was conducted in 49 patients undergoing elective coronary artery bypass graft surgery either with the use of a standard or mini-CPB system (Synergy). We determined early postoperative inflammatory response (leukocytosis, C-reactive protein, urine interleukin-6), platelet consumption and activation (urine thromboxane B2), proximal renal tubular injury (urine N-acetyl-glucosaminidase), and intestinal injury (intestinal fatty acid binding protein). RESULTS: In patients undergoing coronary artery bypass grafting with a mini-CPB system, we observed decreased priming volumes with subsequent attenuation of on-pump hemodilution, improved hemostatic status with reduced platelet consumption and platelet activation, decreased postoperative bleeding and minimized transfusion requirements. We also found reduced leukocytosis and decreased urinary interleukin-6. Levels of urine N-acetyl-glucosaminidase were on average threefold lower, and urinary intestinal fatty acid binding protein was 40% decreased in the patients on the mini-CPB system, as compared with standard CPB. CONCLUSIONS: The use of the mini-CPB system during myocardial revascularization represents a viable nonpharmacologic strategy that can attenuate the alterations in the hemostatic system, reduce bleeding and transfusion requirements, decrease systemic inflammatory response, and reduce immediate postoperative renal and intestinal tissue injury.

Acute isovolemic hemodilution triggers proinflammatory and procoagulatory endothelial activation in vital organs: role of erythrocyte aggregation.

OBJECTIVES: The essential role of erythrocytes as oxygen carriers is historically well established, but their function to aggregate and the consequences on the microcirculation is under debate. The pathogenic potential of low erythrocyte aggregation could be important for patients undergoing on-pump cardipopulmonary bypass. These patients are severely hemodiluted due to preoperative isovolemic hemodilution (IHD), circuit priming, and large fluid infusions perioperatively. Considering the vascular endothelium sensitivity to variations in blood rheology, the authors hypothesize that low erythrocyte aggregation will be responsible for activation of vascular endothelium during acute IHD. METHODS: Acute IHD (30 mL/kg exchange transfusion with colloid solutions) was induced in an "aggregating species"(pigs, n = 15). The hypoxic oxidative stress (plasma malondialdehyde, ex vivo oxygen radicals production in heart, lung, kidney, liver, and ileum tissue biopsies), erythrocyte aggregation (LORCA), and endothelial activation (real-time quantitative RT-PCR on von Willebrand factor (vWF), E- and P-selectins, endothelial nitric oxide synthase gene-expression in tissue biopsies) were investigated. RESULTS: The production of superoxide and hydroxyl radicals, measured as H2O2 generation, was similar at all times in sham-operated and hemodiluted animals, proving a maintained oxygen delivery to tissues. Acute IHD was followed by a dramatic drop in erythrocyte aggregation and immediate prothrombotic (significant vWF mRNA upregulation in heart, lungs, kidney, liver, ileum) and proinflammatory (significant E- and P-selectins mRNA upregulation in lungs and ileum) endothelial activation. Low erythrocyte aggregation was significantly correlated with increased mRNA-expression of vWF (heart, liver, ileum) and P-selectin (lungs, ileum, and heart). CONCLUSIONS: These results suggest that low erythrocyte aggregation might trigger endothelium-dependent thrombogenic and proinflammatory response during acute isovolemic hemodilution.

Dexamethasone: benefit and prejudice for patients undergoing on-pump coronary artery bypass grafting: a study on myocardial, pulmonary, renal, intestinal, and hepatic injury.

STUDY OBJECTIVES: Cardiac surgery with cardiopulmonary bypass (CPB) results in perioperative organ damage caused by the systemic inflammatory response syndrome (SIRS) and ischemia/reperfusion injury. Administration of corticosteroids before CPB has been demonstrated to inhibit the activation of the systemic inflammatory response. However, the clinical benefits of corticosteroid therapy are controversial. This study was designed to document the effects of dexamethasone on cytokine release and perioperative myocardial, pulmonary, renal, intestinal, and hepatic damage, as assessed by specific and sensitive biomarkers. DESIGN AND PATIENTS: A prospective, double-blind, placebo-controlled, randomized trial for dexamethasone was conducted in 20 patients receiving either dexamethasone (1 mg/kg before anesthesia induction and 0.5 mg/kg after 8 h; n = 10) or placebo (n = 10). Different markers were used to assess the SIRS: interleukin (IL)-6, IL-8, IL-10, C-reactive protein (CRP), and tryptase; and organ damage: heart (plasma heart-type fatty acid binding protein, cardiac troponin I [cTnI], creatine kinase-MB), kidneys (N-acetyl-glucosaminidase [NAG], microalbuminuria), intestine (intestinal-type fatty acid binding protein [I-FABP]/liver-type fatty acid binding protein [L-FABP]), and liver (alpha-glutathione S-transferase). RESULTS: Dexamethasone modulated the SIRS with lower proinflammatory (IL-6, IL-8) and higher antiinflammatory (IL-10) IL levels. CRP and tryptase were lower in the dexamethasone group. cTnI values were lower in the dexamethasone group at 6 h in the ICU (p = 0.009). Patients in the dexamethasone group had a longer time to tracheal extubation (18.86 +/- 1.13 h vs 15.01 +/- 0.99 h, p = 0.02 [mean +/- SEM]), with a lower oxygenation index at that time: Pa(O2)/fraction of inspired oxygen ratio, 37.17 +/- 1.8 kPa vs 29.95 +/- 2.1 kPa (p = 0.009). The postoperative glucose level (10.7 +/- 0.6 mmol/L vs 7.4 +/- 0.5 mmol/L, p = 0.005) was higher in the dexamethasone group. Serum glucose was independently associated with intestinal injury (urine I-FABP peak, R2 = 42.5%, beta = 114.4 +/- 31.4, significant at p = 0.002; urine L-FABP peak, R2 = 47.3%, beta = 7,714.1 +/- 1,920.9, significant at p = 0.001) and renal injury (urine NAG, R2 = 32.1%, beta = 0.21 +/- 0.07, significant at p = 0.009). Tryptase peaks correlated negatively with peaks of intestinal and renal injury biomarkers. CONCLUSION: Even while inhibiting SIRS, dexamethasone treatment offered no protection against transient, subclinical, perioperative abdominal organ damage. Tryptase release could have a preconditioning effect, offering protection against perioperative intestinal and renal damage. Dexamethasone treatment resulted in more pronounced postoperative pulmonary dysfunction, prolonged time to tracheal extubation, and initiated postoperative hyperglycemia in patients undergoing elective on-pump coronary artery bypass graft surgery.

Red blood cell aggregation during cardiopulmonary bypass: a pathogenic cofactor in endothelial cell activation?

OBJECTIVE: The bio-incompatibility of the cardiopulmonary bypass (CPB) circuit and the use of artificial colloids trigger massive defense reaction that involves endothelial cells and several blood cells: platelets, neutrophils, monocytes, red blood cells (RBC) and lymphocytes. Investigating the effects on RBC aggregation and endothelial cells activation, the present study addresses two different prime solutions commonly used in the clinical practice. METHODS: RBC aggregation was measured by means of Laser-assisted Optical Rotation Cell Analyzer, in an in vitro study designed to mimic the human blood-material interactions during extracorporeal circulation. A clinical study investigating endothelial activation was conducted in 20 patients undergoing elective coronary bypass surgery, randomly assigned for CPB using two different priming solutions: HAES-steril 6% (HES 200/0.5) and Voluven 6% (HES 130/0.4). RESULTS: Circulation trough a Chandler loop of HES-blood mixes altered significantly RBC aggregability. The use of HES 130/0.4 resulted in marked decrease in RBC aggregation (aggregation index (AI) before and after circulation was 23.5+/-3.8 and 18+/-2.9, respectively), no significant differences being found when compared with Ringer's lactate group. The use of HES 200/0.5 resulted in better maintained RBC aggregation (AI 39.7+/-5.9 and 29.7+/-4.7 before and after circulation, respectively). The AI measured for the whole blood (control) sample was 61.9+/-4.9 before circulation, and 58.1+/-4 after. Markers of endothelial activation (von Willebrand factor (vWF), thrombomodulin (TM), tissue plasminogen activator (tPA) and E-selectin) significantly increased during CPB. Differences between HES treatment groups were evident post-bypass. While the markers of endothelial activation returned to baseline in HES 200/0.5 group, HES 130/0.4 was associated on the first post-operative day with further increase of vWF and tPA. CONCLUSION: RBC aggregation significantly drooped as consequence of blood dilution and blood-material interaction. We reason that low RBC aggregation added to plasma viscosity reduction and non-physiologic flow conditions during extracorporeal circulation are important factors contributing to loss of shear stress at the venous endothelial wall. The loss of shear stress triggers complex signaling leading to endothelial activation. Additional fundamental research is needed in order to verify the hypothesis introduced by the present study. Characterizing the impact of rheologic parameters on endothelial function could prove to be valuable in patients undergoing CPB.

Effect of University of Wisconsin organ-preservation solution on haemorheology.

In conventional cold-storage organ preservation, the donor organ is flushed with University of Wisconsin (UW) solution at 0-4 degrees C. The initial flush is used to wash out blood from the microcirculation to allow optimal preservation with the UW solution. The component hydroxyethyl starch (HES) of UW is known to cause relatively high viscosity and a possible interaction with blood, i.e. increased red blood cell (RBC) aggregation. The aim of this study was to investigate the influence of the HES component on the viscosity of UW and the aggregation behaviour of blood during washout. Viscosity aspects were measured with a cone-plate rheometer. HES-induced RBC aggregation was studied by means of an optical aggregation measuring device. The experiments were carried out with rat whole blood and mixtures of rat whole blood with UW-solution and UW without HES (UWmod), at 4 degrees C. The viscosity of blood at 4 degrees C is two-times higher than at 37 degrees C; the UW/blood mixture at 4 degrees C is 1.3-times more viscous than blood at 37 degrees C; the 4 degrees C UWmod/blood mixture equals the viscosity of blood at 37 degrees C. The UW/blood mixture shows a ninefold increased aggregation compared with whole blood. These aggregates are larger than the diameter of the sinusoids in the rat liver. A mixture of whole blood and UWmod shows a lower aggregation than blood. Apart from an increased viscosity, HES in UW causes increased RBC aggregation. The aggregates are larger than the diameter of the sinusoids. Initial washout could be optimised by pre-flushing to improve the viability of the liver and to decrease delayed graft function.

Hyperaggregating effect of hydroxyethyl starch components and University of Wisconsin solution on human red blood cells: a risk of impaired graft perfusion in organ procurement?

BACKGROUND: The standard preservation solution used during organ procurement and preservation of most organs is the University of Wisconsin (UW) solution. Despite its superiority over other cold storage solutions, the inclusion of hydroxyethyl starch (HES) as one of the components of the UW solution has been both advocated and denied. This study determined whether HES had any effect on red blood cell (RBC) aggregability and correlated aggregation parameters with HES molecular weight. METHODS: Human RBC aggregability and deformability were investigated in vitro, at 4 degrees C, with a laser-assisted optical rotation cell analyzer. The study of RBC aggregation in a binary HES-HES system gave an indication about the nature of HES-RBCs interactions. Bright field microscopy and atomic force microscopy were used to morphologically characterize the aggregates size and form. RESULTS: High molecular weight HES and UW solution had a potent hyperaggregating effect; low molecular weight HES had a hypoaggregating effect on RBC. RBC aggregates were of large size and their resistance to dissociation by flow-induced shear stress was high. CONCLUSION: The authors' in vitro experiments conclusively showed that the physiologic function of RBCs to form aggregates is significantly affected in the presence of HES. The use of high molecular weight HES in UW solution accounts for extended and accelerated aggregation of erythrocytes that may result in stasis of blood and incomplete washout of donor organs before transplantation.

Effect of brain death and non-heart-beating kidney donation on renal function and injury: an assessment in the isolated perfused rat kidney.

OBJECTIVES: Ischemic injury to the renal allograft prior to implantation is considered as the major cause of primary non and never-function (PNF) and delayed graft function (DGF). Evidence has been put forward that brain dead and non-heart-beating (NHB) donor organs are of marginal quality compared to living donors. The purpose of this study was to evaluate renal function and injury of brain dead and NHB donor kidneys using the isolated perfused rat kidney. MATERIAL AND METHODS: Fisher F344 rats were either maintained brain death for 4 hr or subjected to cardiac arrest for 45 min (NHB). Living rats served as controls. To omit additional effects of cold ischemia, kidneys were immediately reperfused. Renal function and injury were assessed by monitoring urine production, glomerular filtration rate (GFR), Na+ and K+ reabsorption, glucose metabolism and reabsorption, as well as release of brush border, lysosomal, and intracellular enzymes. RESULTS: Renal dysfunction and injury were most pronounced in NHB donor kidneys reflected by a highly reduced urine production, anaerobic glucose metabolism resulting in lactate formation, and significant higher luminal release of intracellular and lysosomal enzymes. Brain dead kidneys showed an increased urine production and were functionally abnormal in K+ reabsorption showing a net excretion of K+, probably as a result of ATP depletion. Loss of brush border occurred during brain death and cardiac arrest. CONCLUSIONS: Both, brain death and cardiac arrest have deleterious effects on renal function and renal injury. The ischemically injured NHB donor kidney was functionally inferior compared to the brain dead donor kidney and living donor kidneys. However, both brain dead and NHB kidneys showed considerable renal damage compared to kidneys from living donors.