Claus U. Niemann

Pattern recognition and biomarker validation using quantitative 1H-NMR-based metabolomics

The collection of global metabolic data and their interpretation (both spectral and biochemical) using modern spectroscopic techniques and appropriate statistical approaches, are known as ‘metabolic profiling’, ‘metabonomics’ or ‘metabolomics’. This review addresses 1H-nuclear magnetic resonance (NMR)-based metabolomic principles and their application in biomedical science, with special emphasis on their potential in translational research in transplantation, oncology, and drug toxicity or discovery. Various steps in metabolomics analysis are described in order to illustrate the types of biological samples, their respective handling and preparation for 1H-NMR analysis; provide a rationale for using pattern-recognition techniques (spectral database concept) versus quantitative 1H-NMR-based metabolomics (metabolite database concept); and identify necessary technological and logistical future developments that will allow 1H-NMR-based metabolomics to become an established tool in biomedical research and patient care.

Acute kidney injury during liver transplantation as determined by neutrophil gelatinase‐associated lipocalin

Acute kidney injury (AKI) has significant prognostic implications for long‐term outcomes in patients undergoing liver transplantation. In several retrospective studies, perioperative variables have been associated with AKI. These variables have been mainly associated with changes in creatinine concentrations over several days or months post‐transplantation. To better define AKI, new markers have become available that help to identify patients at risk for renal injury within hours of a triggering insult. We prospectively enrolled liver transplant patients at our institutions to evaluate neutrophil gelatinase‐associated lipocalin (NGAL), a marker of early renal injury, as a surrogate for AKI in patients undergoing liver transplantation. Blood was prospectively collected at predetermined time points from 59 patients at 2 institutions. The electronic anesthesia records and the hospital computer data system were reviewed for perioperative variables. Data collection included patient demographics, intraoperative variables such as fluid management, transfusion requirements, hemodynamics, and urine output. Subsequently, patients were grouped according to the presence of risk for developing AKI as defined by the RIFLE (risk, injury, failure, loss, and end‐stage kidney disease) criteria. The difference between the NGAL concentration 2 hours after reperfusion and the baseline NGAL concentration was predictive of AKI in all patients, including patients with preexisting renal dysfunction. In patients with creatinine concentrations less than 1.5 mg/dL, a single NGAL determination 2 hours after reperfusion of the liver was associated with the development of AKI. Total occlusion of the inferior vena cava was associated with AKI. In conclusion, NGAL concentrations obtained during surgery were highly associated with postoperative AKI in patients undergoing liver transplantation. These findings will allow the design of larger interventional studies. Our findings regarding the impact of surgical techniques and glucose require validation in larger studies. Liver Transpl 15:1852–1860, 2009.

Therapeutic Hypothermia in Deceased Organ Donors and Kidney-Graft Function

BACKGROUND Delayed graft function, which is reported in up to 50% of kidney-transplant recipients, is associated with increased costs and diminished long-term graft function. The effect that targeted mild hypothermia in organ donors before organ recovery has on the rate of delayed graft function is unclear. METHODS We enrolled organ donors (after declaration of death according to neurologic criteria) from two large donation service areas and randomly assigned them to one of two targeted temperature ranges: 34 to 35°C (hypothermia) or 36.5 to 37.5°C (normothermia). Temperature protocols, which were initiated after authorization was obtained for the organ to be donated and for the donors participation in the study, ended when organ donors left the intensive care unit for organ recovery in the operating room. The primary outcome was delayed graft function in the kidney recipients, which was defined as the requirement for dialysis during the first week after transplantation. Secondary outcomes were the rates of individual organs transplanted in each treatment group and the total number of organs transplanted from each donor. RESULTS The study was terminated early, on the recommendation of an independent data and safety monitoring board, after the interim analysis showed efficacy of hypothermia. At trial termination, 370 organ donors had been enrolled (180 in the hypothermia group and 190 in the normothermia group). A total of 572 patients received a kidney transplant (285 kidneys from donors in the hypothermia group and 287 kidneys from donors in the normothermia group). Delayed graft function developed in 79 recipients of kidneys from donors in the hypothermia group (28%) and in 112 recipients of kidneys from donors in the normothermia group (39%) (odds ratio, 0.62; 95% confidence interval, 0.43 to 0.92; P=0.02). CONCLUSIONS Mild hypothermia, as compared with normothermia, in organ donors after declaration of death according to neurologic criteria significantly reduced the rate of delayed graft function among recipients. (Funded by the Health Resources and Services Administration; ClinicalTrials.gov number, NCT01680744.).

Management of the Potential Organ Donor in the ICU: Society of Critical Care Medicine/American College of Chest Physicians/Association of Organ Procurement Organizations Consensus Statement

This document was developed through the collaborative efforts of the Society of Critical Care Medicine, the American College of Chest Physicians, and the Association of Organ Procurement Organizations. Under the auspices of these societies, a multidisciplinary, multi-institutional task force was convened, incorporating expertise in critical care medicine, organ donor management, and transplantation. Members of the task force were divided into 13 subcommittees, each focused on one of the following general or organ-specific areas: death determination using neurologic criteria, donation after circulatory death determination, authorization process, general contraindications to donation, hemodynamic management, endocrine dysfunction and hormone replacement therapy, pediatric donor management, cardiac donation, lung donation, liver donation, kidney donation, small bowel donation, and pancreas donation. Subcommittees were charged with generating a series of management-related questions related to their topic. For each question, subcommittees provided a summary of relevant literature and specific recommendations. The specific recommendations were approved by all members of the task force and then assembled into a complete document. Because the available literature was overwhelmingly comprised of observational studies and case series, representing low-quality evidence, a decision was made that the document would assume the form of a consensus statement rather than a formally graded guideline. The goal of this document is to provide critical care practitioners with essential information and practical recommendations related to management of the potential organ donor, based on the available literature and expert consensus.

Sirolimus, but not the structurally related RAD (everolimus), enhances the negative effects of cyclosporine on mitochondrial metabolism in the rat brain

Clinical studies have shown enhancement of cyclosporine toxicity when co‐administered with the immunosuppressant sirolimus. We evaluated the biochemical mechanisms underlying the sirolimus/cyclosporine interaction on rat brain metabolism using magnetic resonance spectroscopy (MRS) and compared the effects of sirolimus with those of the structurally related RAD. Two‐week‐old rats (25 g) were allocated to the following treatment groups (all n=6): I. control, II. cyclosporine (10 mg kg−1 d−1), III. sirolimus (3 mg kg−1 d−1), IV. RAD (3 mg kg−1 d−1), V. cyclosporine+sirolimus and VI. cyclosporine+RAD. Drugs were administered by oral gavage for 6 days. Twelve hours after the last dose, metabolic changes were assessed in brain tissue extracts using multinuclear MRS. Cyclosporine significantly inhibited mitochondrial glucose metabolism (glutamate: 78±6% of control; GABA: 67±12%; NAD+: 76±3%; P<0.05), but increased lactate production. Sirolimus and RAD inhibited cytosolic glucose metabolism via lactate production (sirolimus: 81±3% of control, RAD: 69±2%; P<0.02). Sirolimus enhanced cyclosporine‐induced inhibition of mitochondrial glucose metabolism (glutamate: 60±4%; GABA: 59±8%; NAD+: 45±5%; P<0.02 versus cyclosporine alone). Lactate production was significantly reduced. In contrast, RAD antagonized the effects of cyclosporine (glutamate, GABA, and NAD+, not significantly different from controls). The results can partially be explained by pharmacokinetic interactions: co‐administration increased the distribution of cyclosporine and sirolimus into brain tissue, while co‐administration with RAD decreased cyclosporine brain tissue concentrations. In addition RAD, but not sirolimus, distributed into brain mitochondria. The combination of cyclosporine/RAD compares favourably to cyclosporine/sirolimus in regards to their effects on brain high‐energy metabolism and tissue distribution in the rat.

Sirolimus delays recovery of rat kidney transplants after ischemia-reperfusion injury.

Background. Sirolimus (SRL) seems to impair renal recovery from ischemic injury in animal models and delayed graft function after clinical renal transplantation. This study determined the impact of SRL on renal recovery after ischemia-reperfusion injury in a rat kidney transplant model. Methods. Syngeneic kidneys were preserved in University of Wisconsin solution before transplantation into bilaterally nephrectomized rats. Recipients received vehicle or SRL targeting for whole-blood trough levels of 10 to 20 ng/mL as measured by high-performance liquid chromatography. Renal function was assessed by animal survival or daily serum creatinine. Tissue samples were collected for histologic examination. Results. Median SRL whole-blood concentrations were 16.6±1.6 ng/mL on postoperative day (POD) 1 and 12.0±0.9 ng/mL on POD 3. Transplantation of kidneys after 39 hr of cold storage resulted in 30% survival in the SRL-treated group compared with 100% survival in the control group (P =0.002). Transplantation of kidneys after 24 hr of cold storage resulted in no survival differences, but there were significant differences in renal function. Daily serum creatinine (PODs 1–4) was higher in the SRL-treated group compared with the control group (P <0.05 at all time points). Grafts from SRL-treated animals showed more severe tubular necrosis compared with control animals. Conclusions. When given at therapeutic immunosuppressive doses, SRL compromises renal function after ischemia-reperfusion injury in a rat kidney transplant model. The antiproliferative effect of SRL may translate into impairment of tubular repair and regeneration necessary for recovery after such injury.

A multicenter evaluation of safety of early extubation in liver transplant recipients

Small single‐institutional studies performed prior to the introduction of organ allocation using the Model for End‐Stage Liver Disease (MELD) suggest that early airway extubation of liver transplant recipients is a safe practice. We designed a multicenter study to examine adverse events associated with early extubation in patients selected for liver transplantation using MELD score. A total of 7 institutions extubated all patients meeting study criteria and reported adverse events that occurred within 72 hours following surgery. Adverse events were uncommon: occurring in only 7.7% of 391 patients studied. Most adverse events were pulmonary or surgically related. Pulmonary complications were usually minor, requiring only an increase in ambient oxygen concentration. The majority of surgical adverse events required additional surgery. Analysis of a limited set of perioperative variables suggest that blood transfusions and technical factors were associated with an increased risk of adverse events. In conclusion, while early extubation appears to be safe under specified circumstances, there are performance differences between institutions that remain to be explained. Liver Transpl 13:1557–1563, 2007.

Transient Hyperglycemia Affects the Extent of Ischemia-Reperfusion-induced Renal Injury in Rats

Background:Chronic hyperglycemia is known to increase renal injury, particularly during ischemia-reperfusion episodes. The goal of this study was to examine whether transient hyperglycemia during or after renal ischemia-reperfusion increased renal dysfunction. Methods:Male Lewis rats underwent sham operations or unilateral nephrectomies followed by contralateral renal ischemia-reperfusion. Hyperglycemic rats were given 25% dextrose to induce transient hyperglycemia lasting throughout the duration of ischemia (PI rats) or beginning 2 h after initiation of reperfusion (PR rats). Additional vehicle control rats received saline and underwent ischemia-reperfusion surgery as with PI and PR rats. Twenty-five minutes of mild renal ischemia followed by 24 h of reperfusion was induced by occluding the renal artery and vein. Results:Terminal serum creatinine concentrations were significantly higher in the PI rats when compared with the PR or vehicle control rats. Histology demonstrated significantly increased necrosis in the PI rats relative to PR and control animals. Tissue analyses demonstrated significantly higher heat shock protein 70, heat shock protein 32, and cleaved caspase-3 protein levels in the PI rats. Oxidative stress generated through the xanthine pathway in the PI group was significantly increased compared with the oxidative stress in the PR and vehicle control rats. In contrast, vascular endothelial growth factor and erythropoietin were significantly decreased in the PI rats compared with the PR rats and controls. Conclusions:Hyperglycemia that occurred during renal ischemia-reperfusion resulted in severe functional injury compared with normoglycemia or with hyperglycemia that occurred after reperfusion. Investigated molecular pathways are more profoundly affected by hyperglycemia that occurs before renal ischemia-reperfusion.

Systemic Inflammation, Coagulopathy, and Acute Renal Insufficiency Following Endovascular Thoracoabdominal Aortic Aneurysm Repair

OBJECTIVE To characterize the inflammatory and coagulopathic response after endovascular thoracoabdominal aortic aneurysm (TAAA) repair and to evaluate the effect of the response on postoperative renal function. METHODS From July 2005 to June 2008, 42 patients underwent elective endovascular repair of a TAAA using custom designed multi-branched stent-grafts at a single academic institution. Four patients were excluded from the analysis. White blood cell count (WBC), platelet count, prothrombin time (PT), and creatinine were measured in all patients. In the last nine patients, interleukin-6 (IL-6), protein C, Factor V, d-dimers, cystatin C, and neutrophil gelatinase-associated lipocalin (NGAL) levels were also measured. Change in lab values were expressed as a percentage of baseline values. RESULTS The 30-day mortality rate was 5% (2/38). All patients (n = 38) had a higher WBC (mean +/- SD: 139 +/- 80%, P < .0001), lower platelet count (56 +/- 15%, P < .0001), and higher PT (median: 17%, Interquartile range (IQR) 12%-22%, P < .0001) after stent-graft insertion. Twelve of 38 patients (32%) developed postoperative acute renal insufficiency (>50% rise in creatinine). Patients with renal insufficiency had significantly larger changes in WBC (178 +/- 100% vs 121 +/- 64%, P = .04) and platelet count (64 +/- 17% vs 52 +/- 12%, P = .02) compared with those without renal insufficiency. All patients (n = 9) had significant increases in NGAL (182 +/- 115%, P = .008) after stent-graft insertion. Six of nine patients (67%) had increased cystatin C (35 +/- 43%, P = .04) after stent-graft insertion, with a greater rise in those with postoperative renal insufficiency (87 +/- 32% vs 8 +/- 13%, P = .02). IL-6 levels were markedly increased in all patients (n = 9) after repair (9840 +/- 6160%, P = .008). Protein C (35 +/- 10%, P = .008) and Factor V levels (28 +/- 20%, P = .008) were uniformly decreased, while d-dimers were elevated after repair in all patients (310 +/- 213%, P = .008). CONCLUSIONS Leukocytosis and thrombocytopenia were uniform following endovascular TAAA repair, and the severity of the response correlated with post-operative renal dysfunction. Elevation of a sensitive marker of renal injury (NGAL) suggests that renal injury may occur in all patients after stent-graft insertion.

Increased severity of renal ischemia-reperfusion injury with venous clamping compared to arterial clamping in a rat model

BACKGROUND Arterial inflow occlusion is a well-known mechanism of renal injury during major vascular surgery. In contrast, renal injury from venous outflow obstruction is poorly understood. The goal of this study was to examine the injury pattern of renal venous outflow obstruction, compare this with the traditional model of arterial occlusion, and examine possible mechanisms. METHODS Male Fisher rats were used for the renal warm ischemia model. Twenty-five minutes of renal ischemia was induced by selectively occluding either the renal artery or vein. After 24 h of reperfusion, whole blood and kidney tissue were collected for further analysis. RESULTS Serum creatinine (SCr) concentrations taken 24 h after reperfusion were significantly greater in the venous occlusion group (V) when compared to the arterial group (A). While histology did not demonstrate significant differences in extent of necrosis between both groups, a stronger inflammatory response resulted from venous occlusion. Specifically, significantly greater MCP-1 mRNA and significantly greater MCP-1, TNF-alpha, and HO-1 protein levels were found in the venous group, while no differences in MIP-2, ICAM-1, and VCAM-1 mRNA expression existed between A and V. Further analysis demonstrated presence of increased cleaved caspase-3 protein in the artery group than in the venous group. CONCLUSIONS Venous renal outflow obstruction results in more severe functional renal injury when compared to arterial inflow occlusion. Macrophage activation and neutrophilic infiltration appear to be exaggerated during venous occlusion.