Neutrophil gelatinase-associated lipocalin does not originate from the kidney during reperfusion in clinical renal transplantation.

BACKGROUND: Acute Kidney Injury (AKI) is a common clinical complication. Plasma/serum neutrophil gelatinase-associated lipocalin (NGAL) has been proposed as a rapid marker of AKI. However, NGAL is not kidney-specific. It exists in three isoforms (monomeric, homo-dimeric and hetero-dimeric). Only the monomeric isoform is produced by renal tubular cells and plasma NGAL levels are confounded by the release of all NGAL isoforms from neutrophils. Our aim was to investigate whether NGAL is released into blood from injured renal tubules. METHODS: Kidney transplantation (n = 28) served as a clinical model of renal ischaemic injury. We used ELISA to measure NGAL concentrations at 2 minutes after kidney graft reperfusion in simultaneously taken samples of renal arterial and renal venous blood. Trans-renal gradients (venous-arterial) of NGAL were calculated. We performed Western blotting to distinguish between renal and non-renal NGAL isoforms. Liver-type fatty acid binding protein (LFABP) and heart-type fatty acid binding protein (HFABP) served as positive controls of proximal and distal tubular damage. RESULTS: Significant renal release of LFABP [trans-renal gradient 8.4 (1.7-30.0) ng/ml, p = 0.005] and HFABP [trans-renal gradient 3.7 (1.1-5.0) ng/ml, p = 0.003] at 2 minutes after renal graft reperfusion indicated proximal and distal tubular damage. NGAL concentrations were comparable in renal venous and renal arterial blood. Thus, there was no trans-renal gradient of NGAL. Western blotting revealed that the renal NGAL isoform represented only 6% of the total NGAL in renal venous blood. CONCLUSIONS: Ischaemic proximal and distal tubular damage occurs in kidney transplantation without concomitant NGAL washout from the kidney graft into blood. Plasma/serum NGAL levels are confounded by the release of NGAL from neutrophils. Present results do not support the interpretation that increase in plasma NGAL is caused by release from the renal tubules.

Endothelial glycocalyx during early reperfusion in patients undergoing cardiac surgery.

BACKGROUND: Experimental cardiac ischemia-reperfusion injury causes degradation of the glycocalyx and coronary washout of its components syndecan-1 and heparan sulfate. Systemic elevation of syndecan-1 and heparan sulfate is well described in cardiac surgery. Still, the events during immediate reperfusion after aortic declamping are unknown both in the systemic and in the coronary circulation. METHODS: In thirty patients undergoing aortic valve replacement, arterial concentrations of syndecan-1 and heparan sulfate were measured immediately before and at one, five and ten minutes after aortic declamping (reperfusion). Parallel blood samples were drawn from the coronary sinus to calculate trans-coronary gradients (coronary sinus-artery). RESULTS: Compared with immediately before aortic declamping, arterial syndecan-1 increased by 18% [253.8 (151.6-372.0) ng/ml vs. 299.1 (172.0-713.7) ng/ml, p < 0.001] but arterial heparan sulfate decreased by 14% [148.1 (135.7-161.7) ng/ml vs. 128.0 (119.0-138.2) ng/ml, p < 0.001] at one minute after aortic declamping. There was no coronary washout of syndecan-1 or heparan sulfate during reperfusion. On the contrary, trans-coronary sequestration of syndecan-1 occurred at five [-12.96 ng/ml (-36.38-5.15), p = 0.007] and at ten minutes [-12.37 ng/ml (-31.80-6.62), p = 0.049] after reperfusion. CONCLUSIONS: Aortic declamping resulted in extracardiac syndecan-1 release and extracardiac heparan sulfate sequestration. Syndecan-1 was sequestered in the coronary circulation during early reperfusion. Glycocalyx has been shown to degrade during cardiac surgery. Besides degradation, glycocalyx has propensity for regeneration. The present results of syndecan-1 and heparan sulfate sequestration may reflect endogenous restoration of the damaged glycocalyx in open heart surgery.

Randomised sham-controlled double-blind trial evaluating remote ischaemic preconditioning in solid organ transplantation: a study protocol for the RIPTRANS trial.

INTRODUCTION: Remote ischaemic preconditioning (RIPC) using a non-invasive pneumatic tourniquet is a potential method for reducing ischaemia-reperfusion injury. RIPC has been extensively studied in animal models and cardiac surgery, but scarcely in solid organ transplantation. RIPC could be an inexpensive and simple method to improve function of transplanted organs. Accordingly, we aim to study whether RIPC performed in brain-dead organ donors improves function and longevity of transplanted organs. METHODS AND ANALYSES: RIPTRANS is a multicentre, sham-controlled, parallel group, randomised superiority trial comparing RIPC intervention versus sham-intervention in brain-dead organ donors scheduled to donate at least one kidney. Recipients of the organs (kidney, liver, pancreas, heart, lungs) from a randomised donor will be included provided that they give written informed consent. The RIPC intervention is performed by inflating a thigh tourniquet to 300 mm Hg 4 times for 5 min. The intervention is done two times: first right after the declaration of brain death and second immediately before transferring the donor to the operating theatre. The sham group receives the tourniquet, but it is not inflated. The primary endpoint is delayed graft function (DGF) in kidney allografts. Secondary endpoints include short-term functional outcomes of transplanted organs, rejections and graft survival in various time points up to 20 years. We aim to show that RIPC reduces the incidence of DGF from 25% to 15%. According to this, the sample size is set to 500 kidney transplant recipients. ETHICS AND DISSEMINATION: This study has been approved by Helsinki University Hospital Ethics Committee and Helsinki University Hospital's Institutional Review Board. The study protocol was be presented at the European Society of Organ Transplantation congress in Copenhagen 14-15 September 2019. The study results will be submitted to an international peer-reviewed scientific journal for publication. TRIAL REGISTRATION NUMBER: NCT03855722.

Graft glycocalyx degradation in human liver transplantation.

OBJECTIVE: Ischaemia/reperfusion-injury degrades endothelial glycocalyx. Graft glycocalyx degradation was studied in human liver transplantation. METHODS: To assess changes within the graft, blood was drawn from portal and hepatic veins in addition to systemic samples in 10 patients. Plasma syndecan-1, heparan sulfate and chondroitin sulfate, were measured with enzyme-linked immunosorbent assay. RESULTS: During reperfusion, syndecan-1 levels were higher in graft caval effluent [3118 (934-6141) ng/ml, P = 0.005] than in portal venous blood [101 (75-121) ng/ml], indicating syndecan-1 release from the graft. Concomitantly, heparan sulfate levels were lower in graft caval effluent [96 (32-129) ng/ml, P = 0.037] than in portal venous blood [112 (98-128) ng/ml], indicating heparan sulfate uptake within the graft. Chondroitin sulfate levels were equal in portal and hepatic venous blood. After reperfusion arterial syndecan-1 levels increased 17-fold (P < 0.001) and heparan sulfate decreased to a third (P < 0.001) towards the end of surgery. CONCLUSION: Syndecan-1 washout from the liver indicates extensive glycocalyx degradation within the graft during reperfusion. Surprisingly, heparan sulfate was taken up by the graft during reperfusion. Corroborating previous experimental reports, this suggests that endogenous heparan sulfate might be utilized within the graft in the repair of damaged glycocalyx.

The origin of plasma neutrophil gelatinase-associated lipocalin in cardiac surgery.

BACKGROUND: Acute kidney injury (AKI) is common after heart surgery. Neutrophil gelatinase-associated lipocalin (NGAL) is produced in injured kidney. NGAL has been used as an early plasma biomarker for AKI in patients undergoing heart surgery. Neutrophils contain all isoforms (25-kDa, 45-kDa and 145-kDa) but the kidney produces almost exclusively the 25-kDa isoform of NGAL. We investigated first, whether there is association between NGAL and neutrophil activation, and second whether activated neutrophils are a significant source of circulating NGAL in plasma in patients undergoing cardiac surgery. METHODS: Two separate patient cohorts were studied: 1) the "kinetic cohort" (n = 29) and 2) the "FINNAKI cohort" (n = 306). As NGAL is strictly co-localized with lactoferrin in neutrophils, NGAL and lactoferrin were measured with enzyme-linked immunosorbent assay in all patients. In sixty-one patients of the "FINNAKI cohort" Western blot was used to separate NGAL isoforms according to their molecular size. Mann-Whitney U, Kruskal-Wallis H, Pearson's and Spearman's tests were used as appropriate. RESULTS: There was strong intraoperative association between NGAL and lactoferrin at all four time-points in the "kinetic cohort". In the "FINNAKI cohort", NGAL and lactoferrin concentrations correlated preoperatively (R = 0.59, p < 0.001) and at admission to the intensive care unit (R = 0.69, p < 0.001). At admission to intensive care unit, concentrations of NGAL and lactoferrin were higher in AKI than in non-AKI patients (NGAL: p < 0.001; lactoferrin: p < 0.029). In Western blot analyses, neutrophil specific 45-kDa isoform (median 41% [IQR 33.3-53.1]) and mostly neutrophil derived 145-kDa isoform (median 53.5% [IQR 44.0-64.9%]) together represented over 90% of total NGAL in plasma. Potentially kidney derived NGAL isoform (25-kDa) accounted for only 0.9% (IQR 0.3 - 3.0%) of total NGAL in plasma. There were no statistically significant differences in the distribution of NGAL isomers between AKI and non-AKI patients. CONCLUSIONS: Plasma NGAL during cardiac surgery is associated with neutrophil activation. Based on molecular size, the majority of circulating NGAL is derived from neutrophils. Neutrophil activation is a confounding factor when interpreting increased plasma NGAL in cardiac surgery.

Glycocalyx Degradation and Inflammation in Cardiac Surgery.

OBJECTIVE: Experimental inflammation induces degradation of glycocalyx. The authors hypothesized that inflammation is an important determinant of glycocalyx degradation in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB). DESIGN: A prospective observational study. SETTING: Operation theater and intensive care unit of a university hospital. PARTICIPANTS: Two separate prospective patient cohorts. INTERVENTIONS: Blood samples were collected at 5 perioperative time points in the trial cohort (30 patients) and only preoperatively in the preoperative cohort (35 patients). Plasma syndecan-1 (biomarker of glycocalyx degradation), interleukin-6 (IL-6), IL-8, and IL-10 were measured. MEASUREMENTS AND MAIN RESULTS: In the trial cohort, preoperative ranges were as follows: 0.8-198 ng/mL for syndecan-1; 0-902 pg/mL for IL-6; 0-314.9 pg/mL for IL-8, and 0-2,909 pg/mL for IL-10. Seven out of 30 patients were outliers in terms of plasma concentrations of syndecan-1 and all cytokines preoperatively. The increase of syndecan-1 was 2.7-fold, and those of IL-6 and IL-8 were both 2.5-fold. The increase of IL-10 was modest. Plasma syndecan-1 correlated with all cytokines preoperatively (IL-6: R = 0.66, p < 0.001; IL-8: R = 0.67, p = 0.001; IL-10: R = 0.73, p < 0.001) as well as at 6 hours postoperatively (IL-6: R = 0.49, p = 0.006; IL-8: R = 0.43, p = 0.02; IL-10: R = 0.41, p = 0.03) and on the postoperative morning (IL-6: R = 0.57, p = 0.001; IL-8: R = 0.37, p = 0.06; IL-10: R = 0.51, p = 0.005) but not intraoperatively. The preoperative findings of the trial cohort could be confirmed in the preoperative cohort. CONCLUSIONS: In patients undergoing cardiac surgery with CPB, inflammation in terms of proinflammatory cytokines IL-6 and IL-8 and anti-inflammatory cytokine IL-10 is associated with glycocalyx degradation measured as plasma syndecan-1 concentrations.

Heparin Binding Protein in Adult Heart Surgery.

BACKGROUND: Heparin binding protein (HBP) is released from neutrophilic secretory vesicles upon neutrophil adhesion on the endothelium. HBP mediates capillary hyperpermeability experimentally. In sepsis, HBP predicts organ dysfunction. Cardiopulmonary bypass induces neutrophil activation and hyperpermeability. We hypothesized that in cardiopulmonary bypass, HBP is released in the reperfused coronary circulation concomitantly with neutrophil adhesion. METHODS: In 30 patients undergoing aortic valve replacement, concomitant blood samples were drawn from the coronary sinus and arterial line before aortic cross-clamping and 5 minutes after reperfusion to calculate transcoronary differences. Plasma HBP concentrations, neutrophil markers lactoferrin and myeloperoxidase, myocardial injury marker heart-type fatty acid binding protein, and leukocyte differential counts were measured. RESULTS: Arterial HBP was 4.1 ng/mL (interquartile range [IQR], 3.6 to 5.3 ng/mL) preoperatively and 150.0 ng/mL (IQR, 108.2 to 188.6 ng/mL) after aortic declamping. HBP increased 39-fold, lactoferrin 16-fold, and myeloperoxidase fourfold during cardiopulmonary bypass. Before cardiopulmonary bypass, there were marginal transcoronary differences in HBP (1.4 ng/mL; IQR, -0.4 to 3.6 ng/mL; p = 0.001) and heart-type fatty acid binding protein (0.4 ng/mL; IQR, -0.04 to 3.5 ng/mL; p = 0.001) but not in the other indicators. During reperfusion, transcoronary HBP release (6.4 ng/mL; IQR, 1.8 to 13.7; ng/mL; p < 0.001) was observed concomitantly with transcoronary neutrophil sequestration (-0.14 × 109/L; IQR, -0.28 to 0.01 × 109/L; p = 0.001) and transcoronary heart-type fatty acid binding protein release (6.9 ng/mL; IQR, 3.0 to 25.8 ng/mL; p < 0.001). There were no transcoronary differences in lactoferrin or myeloperoxidase during reperfusion. CONCLUSIONS: Cardiopulmonary bypass results in substantial increase in circulating HBP. HBP is also released from the reperfused coronary circulation concomitantly with coronary neutrophil adhesion and myocardial injury. HBP may be one candidate for a humoral factor mediating capillary leak in cardiopulmonary bypass.

Heart-Type Fatty Acid Binding Protein and High-Dose Methylprednisolone in Pediatric Cardiac Surgery.

OBJECTIVES: Corticosteroids possess cardioprotection in experimental cardiac ischemia/reperfusion. The authors hypothesized that if cardioprotection of corticosteroids occured during pediatric cardiac surgery, then methylprednisolone used in cardiopulmonary bypass prime would reduce postoperative concentrations of heart-type fatty-acid-binding protein, a cardiac biomarker. DESIGN: A double-blind, placebo-controlled, randomized clinical trial. SETTING: Operating room and pediatric intensive care unit of a university hospital. PARTICIPANTS: Forty-five infants and young children undergoing ventricular or atrioventricular septal defect correction. INTERVENTIONS: The patients received one of the following: 30 mg/kg of methylprednisolone intravenously after anesthesia induction (n = 15), 30 mg/kg of methylprednisolone in cardiopulmonary bypass prime solution (n = 15), or placebo (n = 15). MEASUREMENTS AND MAIN RESULTS: Plasma heart-type fatty-acid-binding protein (hFABP) was measured. Preoperatively, hFABP did not differ among the study groups. Methylprednisolone administered preoperatively and in the cardiopulmonary bypass prime solution reduced hFABP by 44% (p = 0.010) and 38% (p = 0.033) 6 hours postoperatively. hFABP significantly correlated with concomitant troponin T after protamine administration (R = 0.811, p < 0.001) and 6 hours postoperatively (R = 0.806, p < 0.001). CONCLUSIONS: Methylprednisolone in cardiopulmonary bypass prime solution administered only a few minutes before cardiac ischemia confered cardioprotection of the same magnitude as preoperative methylprednisolone as indicated by hFABP concentrations. Rapid cardioprotective actions of corticosteroids in pediatric heart surgery observed previously experimentally may have occurred.