Mild Ischemia-reperfusion Injury Research Articles

Urinary Kidney Injury Biomarkers Are Associated with Ischemia-Reperfusion Injury Severity in Kidney Allograft Recipients.

We explored the potential of emerging and conventional urinary kidney injury biomarkers in recipients of living donor (LD) or donation after circulatory death (DCD) kidney transplantation, patients with chronic kidney disease (CKD), and individuals from the general population. Urine samples from kidney allograft recipients with mild (LD; n = 199) or severe (DCD; n = 71) ischemia-reperfusion injury (IRI) were analyzed for neutrophil gelatinase-associated lipocalin (NGAL), insulin-like growth factor-binding protein 7 (IGFBP7), tissue inhibitor of metalloproteinases 2 (TIMP2), kidney injury molecule-1 (KIM-1), chemokine C-X-C motif (CXCL9), solute carrier family 22 member 2 (SLC22A2), nephrin, and uromodulin (UMOD) by quantitative multiplex LC-MS/MS analysis. The fold-change in biomarker levels was determined in mild and severe IRI and in patients with CKD stage 1-2 (n = 127) or stage ≥3 (n = 132) in comparison to the general population (n = 1438). Relationships between the biomarkers and total protein, β2-microglobulin (B2M), creatinine, and osmolality were assessed. NGAL, IGFBP7, TIMP2, KIM-1, CXCL9, and UMOD were quantifiable, whereas nephrin and SLC22A2 were below the limit of detection. Kidney injury biomarkers were increased up to 6.2-fold in allograft recipients with mild IRI and 8.3-fold in recipients with severe IRI, compared to the reference population, with the strongest response observed for NGAL and B2M. In CKD stage 1-2, B2M, NGAL, IGFBP7, TIMP2, KIM-1, UMOD, and CXCL9 were not altered, but in individuals with CKD stage ≥3, B2M, NGAL, and KIM-1 were increased up to 1.3-fold. IGFBP7, TIMP2, NGAL, and CXCL9 were strongly correlated (all r ≥ 0.8); correlations with B2M and TP were smaller (all r ≤ 0.6). IRI, but not stable CKD, was associated with increased urinary levels of kidney injury biomarkers determined by LC-MS/MS. Absolute and multiplexed protein quantitation by LC-MS/MS is an effective strategy for biomarker panel evaluation for translation toward the clinical laboratory.

In vivo imaging of renal microvasculature in a murine ischemia–reperfusion injury model using optical coherence tomography angiography

Optical coherence tomography angiography (OCTA) provides three-dimensional structural and semiquantitative imaging of microvasculature in vivo. We developed an OCTA imaging protocol for a murine kidney ischemia–reperfusion injury (IRI) model to investigate the correlation between renal microvascular changes and ischemic damage. Mice were divided into mild and moderate IRI groups according to the duration of ischemia (10 and 35 mins, respectively). Each animal was imaged at baseline; during ischemia; and at 1, 15, 30, 45, and 60 mins after ischemia. Amplitude decorrelation OCTA images were constructed with 1.5-, 3.0-, and 5.8-ms interscan times, to calculate the semiquantitative flow index in the superficial (50–70 μm) and the deep (220–340 μm) capillaries of the renal cortex. The mild IRI group showed no significant flow index change in both the superfial and the deep layers. The moderate IRI group showed a significantly decreased flow index from 15 and 45 mins in the superficial and deep layers, respectively. Seven weeks after IRI induction, the moderate IRI group showed lower kidney function and higher collagen deposition than the mild IRI group. OCTA imaging of the murine IRI model revealed changes in superficial blood flow after ischemic injury. A more pronounced decrease in superficial blood flow than in deep blood flow was associated with sustained dysfunction after IRI. Further investigation on post-IRI renal microvascular response using OCTA may improve our understanding of the relationship between the degree of ischemic insult and kidney function.

Suppression of NRF2 Activity by HIF-1α Promotes Fibrosis after Ischemic Acute Kidney Injury.

Acute kidney injury (AKI) is a rapid decline in renal function and can occur after ischemia/reperfusion injury (IRI) to the tubular epithelia. The nuclear factor erythroid-2-related factor 2 (NRF2) pathway protects against AKI and AKI-to-chronic kidney disease (CKD) progression, but we previously demonstrated that severe IRI maladaptively reduced NRF2 activity in mice. To understand the mechanism of this response, we subjected C57BL/6J mice to unilateral kidney IRI with ischemia times that were titrated to induce mild to severe injury. Mild IRI increased NRF2 activity and was associated with renal recovery, whereas severe IRI decreased NRF2 activity and led to progressive CKD. Due to these effects of ischemia, we tested the hypothesis that hypoxia-inducible factor-1α (HIF-1α) mediates NRF2 activity. To mimic mild and severe ischemia, we activated HIF-1α in HK-2 cells in nutrient-replete or nutrient-deficient conditions. HIF-1α activation in nutrient-replete conditions enhanced NRF2 nuclear localization and activity. However, in nutrient-deficient conditions, HIF-1α activation suppressed NRF2 nuclear localization and activity. Nuclear localization was rescued with HIF-1α siRNA knockdown. Our results suggest that severe ischemic AKI leads to HIF-1α-mediated suppression of NRF2, leading to AKI-to-CKD progression.

Postreperfusion Liver Biopsy as Predictor of Early Graft Dysfunction and Survival After Orthotopic Liver Transplantation

Postreperfusion liver biopsy (PRB) can assess the degree of ischemia/reperfusion injury (IRI) after orthotopic liver transplantation (OLT). The influence of IRI on graft outcomes and overall survival is controversial. To determine the correlation between the severity of IRI in PRB and overall graft and patient survival and, secondarily, to identify factors on PRB that predict poor graft outcomes. This is a retrospective analysis of all patients who underwent OLT using donation after brain death (DBD) with PRB. The severity of IRI in PRB was graded. Predictors of IRI were assessed using univariate and multivariate analysis and the Kaplan-Meier with log rank test for the graft and overall survival, respectively. We included 280 OLTs (64.7%). The histopathological assessment of IRI severity was as follows: no IRI (N=96, 34.3%), mild IRI (N=65; 23.2%), moderate IRI (N=101; 36.1%), and severe IRI (N=18; 6.4%). The incidence rates of initial good graft function (IGGF), primary nonfunction and early allograft dysfunction (EAD) were 32.5%, 3.9%, and 18.6%, respectively. Severe IRI was associated with a lower incidence of IGGF (OR: 0.34, 95% CI 0.12-0.92; P=0.03). Patients with severe IRI tended to have a higher incidence of EAD (33.2% vs. 18.6, P=0.23). The cold ischemia time was an independent predictor of severe IRI on the multivariate analysis. Severe IRI was associated with poor 1- and 5-year overall survival rates (67% and 44%, respectively, compared with 84 and 68% in nonsevere IRI). Patients with severe IRI exhibited worse graft and overall survival. Cold ischemia time predicts the development of severe IRI. Patients with severe IRI show worse graft and overall survival and a lower incidence of IGGF, suggesting that histopathological findings could be useful for identifying patients at high risk of worse outcomes after OLT.

Spontaneous, transient adenosine release is not enhanced in the CA1 region of hippocampus during severe ischemia models.

Ischemic stroke causes damage in the brain, and a slow buildup of adenosine is neuroprotective during ischemic injury. Spontaneous, transient adenosine signaling, lasting only 3s per event, has been discovered that increases in frequency in the caudate-putamen during early stages of mild ischemia-reperfusion injury. However, spontaneous adenosine changes have not been studied in the hippocampus during ischemia, an area highly susceptible to stroke. Here, we investigated changes of spontaneous, transient adenosine in the CA1 region of rat hippocampus during three different models of the varied intensity of ischemia. During the early stages of the milder bilateral common carotid artery occlusion (BCCAO) model, there were fewer spontaneous, transient adenosine, but no change in the concentration of individual events. In contrast, during the moderate 2 vertebral artery occlusion (2VAO) and severe 4 vessel occlusion (4VO) models, both the frequency of spontaneous, transient adenosine and the average event adenosine concentration decreased. Blood flow measurements validate that the ischemia models decreased blood flow, and corresponding pathological changes were observed by transmission electron microscopy (TEM). 4VO occlusion showed the most severe damage in histology and BCCAO showed the least. Overall, our data suggest that there is no enhanced spontaneous adenosine release in the hippocampus during moderate and severe ischemia, which could be due to depletion of the rapidly releasable adenosine pool. Thus, during ischemic stroke, there are fewer spontaneous adenosine events that could inhibit neurotransmission, which might lead to more damage and less neuroprotection in the hippocampus CA1 region. Read the Editorial Highlight for this article on page 800.

NLRX1 Deletion Increases Ischemia-Reperfusion Damage and Activates Glucose Metabolism in Mouse Heart.

BackgroundNOD-like receptors (NLR) are intracellular sensors of the innate immune system, with the NLRP3 being a pro-inflammatory member that modulates cardiac ischemia-reperfusion injury (IRI) and metabolism. No information is available on a possible role of anti-inflammatory NLRs on IRI and metabolism in the intact heart. Here we hypothesize that the constitutively expressed, anti-inflammatory mitochondrial NLRX1, affects IRI and metabolism of the isolated mouse heart.MethodsIsolated C57Bl/6J and NLRX1 knock-out (KO) mouse hearts were perfused with a physiological mixture of the essential substrates (lactate, glucose, pyruvate, fatty acid, glutamine) and insulin. For the IRI studies, hearts were subjected to either mild (20 min) or severe (35 min) ischemia and IRI was determined at 60 min reperfusion. Inflammatory mediators (IL-6, TNFα) and survival pathways (mito-HKII, p-Akt, p-AMPK, p-STAT3) were analyzed at 5 min of reperfusion. For the metabolism studies, hearts were perfused for 35 min with either 5.5 mM 13C-glucose or 0.4 mM 13C-palmitate under normoxic conditions, followed by LC-MS analysis and integrated, stepwise, mass-isotopomeric flux analysis (MIMOSA).ResultsNLRX1 KO significantly increased IRI (infarct size from 63% to 73%, end-diastolic pressure from 59 mmHg to 75 mmHg, and rate-pressure-product recovery from 15% to 6%), following severe, but not mild, ischemia. The increased IRI in NLRX1 KO hearts was associated with depressed Akt signaling at early reperfusion; other survival pathways or inflammatory parameters were not affected. Metabolically, NLRX1 KO hearts displayed increased lactate production and glucose oxidation relative to fatty acid oxidation, associated with increased pyruvate dehydrogenase flux and 10% higher cardiac oxygen consumption.ConclusionDeletion of the mitochondrially-located NOD-like sensor NLRX1 exacerbates severe cardiac IR injury, possibly through impaired Akt signaling, and increases cardiac glucose metabolism.

EXTRACORPOREAL MEMBRANE SUPPORT AS A BRIDGE TO PULMONARY THROMBOENDARTERECTOMY IN CHRONIC THROMBOEMBOLIC PULMONARY HYPERTENSION: A NOVEL STRATEGY IN PULMONARY ISCHEMIA-REPERFUSION INJURY

SESSION TITLE: Case Report Semifinalists 5 SESSION TYPE: Fellow Case Report Posters PRESENTED ON: 10/21/2019 02:30 PM - 03:15 PM INTRODUCTION: The description of Ischemia-reperfusion injury (IRI) is first accredited to Dr. R. B. Jennings’ in his 1960 animal study on myocardial necrosis. IRI is initiated by an ischemic or inflammatory injury to the alveolar capillary endothelium activating NADPH oxidase 3 (NOX-3) and inducible Nitric Oxide Synthase (iNOS). Reperfusion and hyperoxia provide substrate for these oxidation reactions to create an oxidative stress environment. IRI can be associated with significant morbidity and mortality following lung transplantation and pulmonary thromboendarterectomy (PTE). We describe a unique case of acute lung injury complicated CTEPH for which V-V ECMO was used as a bridge to PTE to limit IRI. CASE PRESENTATION: A 43-year-old female with known CTEPH presented to an outside hospital with progressive dyspnea, hypoxia and right sided weakness. CT head disclosed left occipital lobe and cerebellar infarcts. Chest CTA showed a chronic complete occlusion of the left pulmonary trunk and acute right segmental and subsegmental thrombi (level 1). An echocardiogram disclosed a PFO and severe right ventricular enlargement. Right heart catheterization (RHC) disclosed severe pulmonary hypertension (mPA 59, wedge 11). A mechanical thrombectomy was unsuccessfully attempted. Patient was transferred for emergent PTE evaluation, however, developed acute hypoxic respiratory failure and was intubated upon arrival. Repeat CT chest disclosed dense right > left organizing infiltrates bibasilar suggestive of acute lung injury. Due to high risk of IRI, PTE was deferred, and patient was bridged to V-V EMCO for ongoing intractable hypoxemia and acute lung injury. I.V. epoprostenol was initiated for medical management for PH. She was decannulated after 2 weeks, however, required intermitted mechanical ventilation and aggressive physical therapy. PTE was successfully performed on hospital day 54 and epoprostenol was immediately discontinued. One-time doses of Lasix 20mg and solumedrol 125mg were given for mild IRI. Tracheostomy was decannulated by postop day 11 and patient was discharged home without oxygen. DISCUSSION: IRI creates tissue injury via superoxide species, capillary hyperpermeability, and activation of innate immune system via downstream proinflammatory chemokines. We present a unique case of acute lung injury in the setting of CTEPH requiring PTE. We utilized V-V ECMO as a bridge to PTE surgery resulting in a two-step strategy to limit IRI:(1)Allowance of adequate recovery from the initial endothelial injury employing supportive measures not excluding pulmonary/circulatory support if necessary;(2)Early proactive management of the inflammatory and capillary leak sequela of IRI as expected known entities after reperfusion. CONCLUSIONS: This strategy allows repletion of antioxidants. Further investigatory studies on this topic will hopefully provide clarity on the efficacy of this strategy. Reference #1: De Perrot M, Liu, M, Waddell TK, Keshavjee S. Ischemia-reperfusion-Induced Lung Injury. American Journal of Respiratory and Critical Care Medicine. 2003;167(4):490-511 Reference #2: Jennings RB, Sommers HM, Smyth GA, Flack HA, Linn H. Myocardial necrosis induced by temporary occlusion of a coronary artery in the dog. Arch Pathol. 1960;70:68–78 Reference #3: Ovechkin AV, Lominadze D, Sedoris KC, Robinson TW, Tyagi SC, Roberts AM. Lung ischemia-reperfusion injury: implications of oxidative stress and platelet-arteriolar wall interactions. Arch Physiol Biochem. 2007;113(1):1-12. DISCLOSURES: No relevant relationships by Tony Hodges, source=Web Response No relevant relationships by Kurt Olson, source=Web Response No relevant relationships by Esa Rayyan, source=Web Response No relevant relationships by Rajeev Saggar, source=Web Response No relevant relationships by Sarika Savajiyani, source=Web Response No relevant relationships by Nafis Shamsid-Deen, source=Web Response No relevant relationships by Gurvijay Singh, source=Web Response No relevant relationships by Ramachandra Sista, source=Web Response No relevant relationships by Steve Tseng, source=Web Response

Overexpression of Human CD55 and CD59 or Treatment with Human CD55 Protects against Renal Ischemia-Reperfusion Injury in Mice.

Deficiency in the membrane-bound complement regulators CD55 and CD59 exacerbates renal ischemia-reperfusion injury (IRI) in mouse models, but the effect of increasing CD55 and CD59 activity has not been examined. In this study, we investigated the impact of overexpression of human (h) CD55 ± hCD59 or treatment with soluble rhCD55 in a mouse model of renal IRI. Unilaterally nephrectomised mice were subjected to 18 (mild IRI) or 22 min (moderate IRI) warm renal ischemia, and analyzed 24 h after reperfusion for renal function (serum creatinine and urea), complement deposition (C3b/c and C9), and infiltration of neutrophils and macrophages. Transgenic mice expressing hCD55 alone were protected against mild renal IRI, with reduced creatinine and urea levels compared with wild type littermates. However, the renal function of the hCD55 mice was not preserved in the moderate IRI model, despite a reduction in C3b/c and C9 deposition and innate cell infiltration. Mice expressing both hCD55 and hCD59, on the other hand, were protected in the moderate IRI model, with significant reductions in all parameters measured. Wild type mice treated with rhCD55 immediately after reperfusion were also protected in the moderate IRI model. Thus, manipulation of CD55 activity to increase inhibition of the C3 and C5 convertases is protective against renal IRI, and the additional expression of hCD59, which regulates the terminal complement pathway, provides further protection. Therefore, anti-complement therapy using complement regulatory proteins may provide a potential clinical option for preventing tissue and organ damage in renal IRI.

Negligible Oval Cell Proliferation Following Ischemia-Reperfusion Injury With and Without Partial Hepatectomy.

Hepatic oval cells proliferate to replace hepatocytes and restore liver function when hepatocyte proliferation is compromised or inadequate. Exposure to chemical carcinogens, severe liver steatosis, and partial hepatectomy has been used in animal models to demonstrate the role of oval cells in liver regeneration. Ischemia-reperfusion injury (IRI) causes hepatocellular damage and death in the absence of confounding chemical toxicity; however, oval cell induction by IRI has not been demonstrated in vivo. We examine oval cell induction following partial IRI. Wistar rats were subjected to 2 IRI protocols: 70% warm liver ischemia for 30 minutes followed by reperfusion or 70% warm liver ischemia for 30 minutes with partial hepatectomy of the nonischemic lobes followed by reperfusion. Liver injury was monitored by serum alanine aminotransferase (ALT) at 1 day and 7 days of reperfusion. Oval cell proliferation was monitored by indirect immunofluorescence staining using the surface markers BD.2 and Thy-1. Cellular proliferation was quantified by 5-ethynyl-2'-deoxyuridine (EdU) incorporation in vivo. Serum ALT elevation was only observed at the 1-day time point in the IRI with partial hepatectomy model. Oval cell marker expression was restricted to the biliary structures in both the ischemic and the nonischemic control lobes. Oval cell induction, measured by changes in the frequency of BD.2 and Thy-1 expression and EdU incorporation, was not significantly altered by IRI. In both mild and moderate IRI models, we did not find evidence of oval cell induction or proliferation. EdU staining was restricted to hepatocytes, suggesting that liver regeneration following IRI is mediated by hepatocyte proliferation.

Both PD-1 ligands protect the kidney from ischemia reperfusion injury.

Acute kidney injury (AKI) is a common problem in hospitalized patients that enhances morbidity and mortality and promotes the development of chronic and end-stage renal disease. Ischemia reperfusion injury (IRI) is one of the major causes of AKI and is characterized by uncontrolled renal inflammation and tubular epithelial cell death. Our recent studies demonstrated that regulatory T cells (Tregs) protect the kidney from ischemia reperfusion-induced inflammation and injury. Blockade of programmed death-1 (PD-1) on the surface of Tregs, prior to adoptive transfer, negates their ability to protect against ischemic kidney injury. The present study was designed to investigate the role of the known PD-1 ligands, PD-L1 and PD-L2, in kidney IRI. Administration of PD-L1 or PD-L2 blocking Abs prior to mild or moderate kidney IRI significantly exacerbated the loss of renal function, renal inflammation, and acute tubular necrosis compared with mice receiving isotype control Abs. Interestingly, blockade of both PD-1 ligands resulted in worse injury, dysfunction, and inflammation than did blocking either ligand alone. Genetic deficiency of either PD-1 ligand also exacerbated kidney dysfunction and acute tubular necrosis after subthreshold ischemia. Bone marrow chimeric studies revealed that PD-L1 expressed on non-bone marrow-derived cells is critical for this resistance to IRI. Finally, blockade of either PD-1 ligand negated the protective ability of adoptively transferred Tregs in IRI. These findings suggest that PD-L1 and PD-L2 are nonredundant aspects of the natural protective response to ischemic injury and may be novel therapeutic targets for AKI.

An innovative model of autologous anorectal transplantation with pudendal nerve reconstruction

Fecal incontinence is an important public health problem that has a significant impact on the quality of life of individual patients. The prevalence of fecal incontinence in the United States ranges from 2 to 17%, but this may be an underestimation (1). The current treatments for severe fecal incontinence are often complex and have disappointing results (1). Anorectal transplantation may be a definitive method for restoring normal sphincter function in these patients. We and others have previously investigated anorectal transplantation in rats with promising results (2-4). However, there is a lack of pre-clinical large animal models to study the potential benefits of this procedure. In this paper, we describe an innovative surgical technique of anorectal autotransplantation, which allows for physiological studies without the immunologic effects related to allotransplantation. The technique was initially performed in three swine weighing 25-35 Kg. After general anesthesia, an abdominal and perineal incision was performed in which the rectum, anal sphincter, and skin surrounding the anus were procured en bloc. The graft's vascular pedicle contained the inferior mesenteric artery (IMA) with a patch of the aorta and inferior mesenteric vein (IMV), which was divided near the splenic vein (Figure 1A). The pudendal nerve was carefully isolated and transected. The graft was removed and immediately flushed with 250 ml of cold heparinized preservation solution through a catheter inserted in the IMA. The graft was then placed in a cooler with cold solution without heparin for a total of 20 minutes. The infrarenal aorta and vena cava were isolated and cross-clamped, and the graft was implanted by arterial and venous anastomoses between the IMA and the aorta (Figure 1B) and the IMV and vena cava (Figure 1C). After reperfusion (Figure 1D), the entire graft was placed inside the abdominal cavity, and the pudendal nerve was reconstructed (Figure 1E). Finally, the anus was placed in its original position, and the perineum was closed (Figure 1F). Digestive tract continuity was reestablished by an end-to-end colorectal anastomosis. The abdominal cavity was closed, and the animals were observed for an additional two hours. At the end of the experiment, the graft was removed for histological analysis. The average times for graft recovery and implantation (including vascular, nervous and GI reconstruction) were 57 and 99 minutes, respectively. No problems regarding graft perfusion were observed at the end of the experiment. Histological graft assessment showed normal aspects in one graft and mild ischemia-reperfusion injury in two. Figure 1 (A) An anorectal graft showing the inferior mesenteric artery (IMA) and vein (IMV) and the rectum (R). (B) The infrarenal aorta (Ao) and vena cava (IVC) were cross-clamped at the site of anastomosis. (C) The anastomoses between the Ao and IMA and between ... To the best of our knowledge, there is only one report of a pre-clinical model of anorectal transplantation (5). Those authors described the donor and recipient surgical technique in four swine allotransplantations, with a 25% rate of technical failure after a 24-hour observation period. The experimental model described in this paper mimics the surgical stress and potential effects of graft denervation of allotransplantation but precludes, for obvious reasons, the antigen-mediated immune response. In addition, the technical modifications were associated with a significant reduction in the surgical time and cost. Therefore, anorectal autotransplantation in swine is a feasible and relatively simple procedure that may be a relevant technique to study regeneration, anorectal physiology, and the potential benefits of this unique type of transplantation without the interference of immune-mediated effects.

Hepatic blood flow plays an important role in ischemia-reperfusion injury

Severe ischemia/reperfusion (IR) injury is associated with poor hepatic microperfusion. The aim of this study was to investigate the role of hepatic artery flow (HAF) and portal vein flow (PVF) in IR injury. From January 2004 to June 2008, 566 patients underwent orthotopic liver transplantation (OLT). The data were retrospectively reviewed via the transplant database. Patients with hepatic artery (HA) or portal vein (PV) thrombosis and retransplant patients were excluded. Intraoperative PVF and HAF values and graft weights were measured routinely, and the central venous pressure, mean arterial pressure, cardiac output, and cardiac index were recorded with hepatic blood flow measurements. Complete data were available for 312 primary OLT recipients (215 males and 97 females; mean age = 54 ± 10 years). The patients' follow-up ranged from 215 to 1746 days (705 ± 408 days). IR injury was defined by the aspartate aminotransferase (AST) level on postoperative day (POD) 2, and the patients were divided into 3 groups: (1) mild IR injury [AST < 500 U/L; n = 160 (51%)], (2) moderate IR injury [AST = 500-1000 U/L; n = 85 (27%)], and (3) severe IR injury [AST > 1000 U/L; n = 67 (21%)]. The demographics and pre-OLT variables (the Model for End-Stage Liver Disease score (MELD), platelet counts, PV thrombosis, transjugular intrahepatic portosystemic shunts, and shunts on computed tomography scans) were similar in all groups. The graft survival rate was 99% in group 1, 95.2% in group 2 (P = 0.02), and 92.3% in group 3 (P = 0.016). The patient survival rates were similar in the 3 groups. The cold ischemia time (CIT) was significantly higher in group 3 versus group 1 (P < 0.007). In the statistical analysis, low HAF, PVF, total liver blood flow (TLBF), and augmented HAF values were associated with a greater likelihood of elevated AST levels on POD 2. The strongest univariate predictors of AST were reduced augmented HAF (mL/minute/100 g) values (P < 0.001) and reduced TLBF (mL/minute/100 g) values (P < 0.001). In a covariate analysis with adjustments for CIT and donor variables, the blood flow parameters remained important predictors of graft function. In conclusion, this report demonstrates for the first time that reduced hepatic blood flow is a significant finding in patients with severe hepatic IR injury.

Diannexin Protects against Renal Ischemia Reperfusion Injury and Targets Phosphatidylserines in Ischemic Tissue

Renal ischemia/reperfusion injury (IRI) frequently complicates shock, renal transplantation and cardiac and aortic surgery, and has prognostic significance. The translocation of phosphatidylserines to cell surfaces is an important pro-inflammatory signal for cell-stress after IRI. We hypothesized that shielding of exposed phosphatidylserines by the annexin A5 (ANXA5) homodimer Diannexin protects against renal IRI. Protective effects of Diannexin on the kidney were studied in a mouse model of mild renal IRI. Diannexin treatment before renal IRI decreased proximal tubule damage and leukocyte influx, decreased transcription and expression of renal injury markers Neutrophil Gelatinase Associated Lipocalin and Kidney Injury Molecule-1 and improved renal function. A mouse model of ischemic hind limb exercise was used to assess Diannexin biodistribution and targeting. When comparing its biodistribution and elimination to ANXA5, Diannexin was found to have a distinct distribution pattern and longer blood half-life. Diannexin targeted specifically to the ischemic muscle and its affinity exceeded that of ANXA5. Targeting of both proteins was inhibited by pre-treatment with unlabeled ANXA5, suggesting that Diannexin targets specifically to ischemic tissues via phosphatidylserine-binding. This study emphasizes the importance of phosphatidylserine translocation in the pathophysiology of IRI. We show for the first time that Diannexin protects against renal IRI, making it a promising therapeutic tool to prevent IRI in a clinical setting. Our results indicate that Diannexin is a potential new imaging agent for the study of phosphatidylserine-exposing organs in vivo.

Endotoxin tolerance does not limit mild ischemia-reperfusion injury in humans in vivo

Animal studies have shown that previous exposure to lipopolysaccharide (LPS) can limit ischemia-reperfusion injury. We tested whether pretreatment with LPS also protects against ischemia-reperfusion injury in humans in vivo. Fourteen volunteers received bolus injections of incremental dosages of LPS on 5 consecutive days (LPS group). Before the first and 1 day after the last LPS administration, the forearm circulation of the non-dominant arm was occluded for 10 min, with concomitant intermittent handgripping to induce transient ischemia. After reperfusion, 0.1 mg of ( 99m)Tc-labeled annexin A5 (400 MBq) was injected intravenously to detect phosphatidylserine expression as an early marker of ischemia-reperfusion injury. Similarly, the control group (n = 10) underwent the ischemic exercise twice, but without pretreatment with LPS. Annexin A5 targeting was expressed as the percentage difference in radioactivity in the thenar muscle between both hands. Endotoxin tolerance developed during 5 consecutive days of LPS administration. Annexin A5 targeting was 12.1 +/- 2.2% and 10.4 +/- 2.1% before LPS treatment at 1 h and 4 h after reperfusion, compared to 12.2 +/- 2.4% and 8.9 +/- 2.1% at 1 h and 4 h after reperfusion on day 5 (P = 1.0 and 0.6, respectively). Also, no significant changes in annexin A5 targeting were found in the control group. So, in this model, LPS-tolerance does not protect against ischemia-reperfusion injury in humans in vivo.

Jaundice developing in a patient with hepatitis C 4 months after liver transplantation

A 52-year-old man underwent liver transplantation (LT) for hepatitis C virus (HCV)-related cirrhosis. Before LT, he had a Model for End-Stage Liver Disease score of 32 and had been hospitalized with spontaneous bacterial peritonitis and hepatorenal syndrome requiring hemodialysis. He was infected with genotype 1A with a viral load of 1,800,000 IU and previously did not respond to interferon. He received a liver from a 66-year-old donor with 9 hours of cold ischemia time. The liver suffered mild ischemia-reperfusion injury and the post-LT course was complicated by renal failure requiring dialysis. Steroid-free immunosuppression was used with interleukin-2 antagonist induction, mycophenolate, and tacrolimus. All liver tests normalized, and the patient was discharged after 3 weeks. ALT, alanine aminotransferase; AST, aspartate aminotransferase; HCV, hepatitis C virus; LT, liver transplantation. Tacrolimus trough levels were maintained at 10-12 μg/L with gradual normalization of renal function. Liver tests remained normal but the ascites never totally resolved. The patient became jaundiced 4 months after LT with aspartate aminotransferase (AST) 110 U/L, alanine aminotransferase (ALT) 127 U/L, alkaline phosphatase 390 U/L, and direct bilirubin 6.2 mg/dL. Doppler ultrasonography showed no ductal dilatation and the hepatic artery was patent. Magnetic resonance imaging cholangiography showed no evidence of biliary stricture. He was afebrile with hemoglobin 11.6 g/dL, platelets 80,000/μL, and creatinine 2.1 mg/dL. Cytomegalovirus was undetectable by polymerase chain reaction and HCV RNA 18,500,000 IU. A liver biopsy was performed and showed the following: Low-power magnification showing cholestasis with feathery degeneration of hepatocytes (arrows) and minimal inflammation is seen in portal areas (Fig. A). A ductular reaction is noted (arrow heads). Trichrome stain of one representative portal tract shows fibrous tissue and delicate fibrous septa extending into periportal sinusoids and occasionally enclosing hepatocytes (arrows; Fig. B). These features are consistent with the cholestatic variant of recurrent HCV.1 Treatment with pegylated interferon-α2a and ribavirin was initiated, but the jaundice and ascites worsened. The AST, ALT, and alkaline phosphatase decreased minimally and the direct bilirubin increased to 17.1 mg/dL. The HCV RNA decreased to 1,500,000 IU 6 weeks into treatment, but the patient developed gradual renal failure and severe anemia requiring cessation of antiviral treatment; he soon developed enterococcal bacteremia and died. The cholestatic variant of recurrent HCV is typified by cholestatic liver tests and an extremely high HCV viral load occurring early after LT. It occurs infrequently and generally results in graft failure, carrying a high mortality rate.1 Its histological findings are in contradistinction to typical acute recurrent HCV (Fig. C) in which there is more necroinflammation, absence of cholestasis, and less fibrosis. Acidophilic bodies (arrowheads), lobular inflammatory infiltrates, and spotty necrosis are seen. Response to interferon therapy is poor, and retransplantation may be required. Its pathogenesis is poorly understood with risk factors being older donor age, significant HCV viremia, and strong immunosuppression.2 It has also been described in human immunodeficiency virus, after renal and cardiac transplantation, and after chemotherapy in patients with HCV or HBV.3, 4 A similar histologic syndrome is well-described in patients with recurrent HBV after LT.5 The early post-LT differential diagnosis of abnormal liver tests in this patient included drug hepatotoxicity, biliary obstruction or hepatic artery thrombosis, cytomegalovirus infection, acute/chronic rejection, as well as recurrent HCV. Definitive histopathologic diagnosis of cholestatic HCV recurrence may be problematic because more than one of the above processes may be present in the same biopsy. Appropriate exclusion of other diagnoses and provision of clinical and laboratory information to the pathologist is essential.

Mild renal ischemia-reperfusion reduces charge and size selectivity of the glomerular barrier

Despite recent discoveries of molecules in podocytes, the mechanisms behind most conditions of proteinuria are still poorly understood. To understand more about this delicate barrier, we studied the functional and morphological effects of mild (15 min) renal ischemia-reperfusion injury (IRI). Renal function was studied in rats in vivo, followed by a more detailed analysis of the glomerular barrier in cooled (8 degrees C) isolated perfused kidneys (cIPK). Renal blood flow was quickly restored, whereas the glomerular filtration rate remained halved 30 min after IRI. Tubular cell activity was intact as judged from the unaffected Cr-EDTA U/P concentration ratio. In vivo, the fractional clearance (theta) for albumin increased 16 times. In rats subjected to cIPK starting 30 min after in vivo IRI, theta(albumin) was 15 times and theta(Ficoll_36angstroms) 1.8 times higher than in control cIPKs. According to the heterogeneous charged fiber model, IRI reduced the fiber charge density to 38% of control (P < 0.01, n = 7). Morphometric analysis with electron microscopy did not reveal any changes in the podocytes or the glomerular basement membrane (GBM) after IRI, suggesting more subtle changes of the GBM and/or the endothelial glycocalyx. We conclude that mild renal IRI induces formation of reactive oxygen species, massive proteinuria, and loss of charged fibers with no apparent change in morphology. These novel findings stress the importance of other components of the barrier, such as proteoglycans produced by the glomerular cells, and provide a tentative explanation for the mechanisms behind proteinuria in glomerulonephritis, for example.

Safety and Efficacy of a Novel Gel for Vascular Occlusion in Off-Pump Surgery

Coronary occlusion techniques used during off-pump coronary artery bypass surgery often provide suboptimal visualization and can damage the endothelium. We evaluated a novel gel with reverse thermosensitive properties for internal vessel occlusion during off-pump coronary artery bypass surgery. Yorkshire pigs (n = 6 per group) underwent two cycles of mid-left anterior descending coronary artery (LAD) occlusion using the gel (injected into the artery) or microvascular clamps (control group) followed by 30 minutes of reperfusion. Regional wall motion and LAD flow were monitored, microvessel relaxation responses were evaluated, and myocardial tissue was analyzed histologically. Complete left anterior descending coronary artery occlusion was successfully achieved using the gel (median ischemic time, 14 minutes; range, 4.5 to 24 minutes). Anterior wall motion abnormalities as well as flow patterns in the reperfused left anterior descending coronary artery were similar in both groups. Microvessel relaxation to substance P was mildly impaired (-11.7 +/- 2.8% vs control; p < 0.001) in the left anterior descending coronary artery territory, but response to adenosine diphosphate and sodium nitroprusside was unaffected. Mild contraction band necrosis was present in both groups, consistent with mild ischemia-reperfusion injury. The gel represents a safe and effective method of vessel occlusion with a potentially important role in off-pump coronary artery bypass surgery.

Bacterial Translocation of Coagulase-Negative Staphylococci in Rats following Intestinal Ischemia-Reperfusion Injury

Many patients with sepsis have bacteremia for which no septic focus is identified either clinically or by autopsy. This study was designed to determine the relationship between the ischemia-reperfusion injury (IRI) and bacterial translocation that might be involved in the pathogenesis of necrotizing enterocolitis. In the first experiment, a total of 32 Sprague-Dawley rats weighing 150–200 g were divided into four groups. The mesentery to isolated loop was occluded for 30, 60, and 90 min following 30-min reperfusion in the three groups of experimental animals with a micro-bulldog clamp. A control group involved the same technique and exposure, without occlusion of the mesentery. Two sets of blood culture were taken through a catheter in the portal vein immediately and 15 min after the reperfusion, respectively. In another experiment, bacteria isolated were fed in different doses to control rats and those after 30- or 60-min ischemia and 30-min reperfusion. Two sets of blood culture were taken following the procedure. Invasion and transcytosis of the bacteria through epithelial cells were studied in vitro using a Madin-Derby canine kidney (MDCK) cell monolayer model. PCR for delta toxin gene was performed on all bacteria isolated, using Staphylococcus epidermidis as the control. Coagulase-negative staphylococci (CoNS) were invariably isolated from mice with prolonged ischemia (90 min) and reperfusion. When bacteria were fed into mice with only 30-min ischemia, an inoculum as low as 5 × 10⁵ CFU/ml could induce bacteremia. No bacterial translocation was found in control mice even fed with a higher dose of bacteria (5 × 10⁸ CFU/ml). In vitro experiments showed that CoNS failed to transcytose MDCK monolayer. These isolates were not cytotoxic to MDCK cells and contained no delta toxin gene. Bacterial translocation of CoNS occurred following severe bowel ischemia and reperfusion injury. Intact mucosa integrity readily prevented bacterial translocation; however, bacterial translocation could occur in rats following mild IRI in the presence of a higher number of CoNS in the gut.

Protective effect of glycine on renal injury induced by ischemia-reperfusion in vivo.

Although glycine prevents renal tubular cell injury in vitro, its effect in vivo is not clear. The purpose of this study was to investigate whether a bolus injection of glycine given before reperfusion plus continuous dietary supplementation afterward would reduce renal injury caused by ischemia-reperfusion. Female Sprague-Dawley rats received a semisynthetic powdered diet containing 5% glycine and 15% casein (glycine group) or 20% casein (control group). Two days later, renal ischemia was produced by cross-clamping the left renal vessels for 15 min, followed by reperfusion. The right kidney was removed before reperfusion. The postischemic glomerular filtration rate (GFR) showed that renal function was less impaired and recovered more quickly in rats receiving glycine. For example, at day 7, GFR in controls (0.31 +/- 0.03 ml x min(-1) x 100 g(-1)) was about one-half that of glycine-treated rats (0.61 +/- 0.06 ml x min(-1) x 100 g(-1), P < 0.05). Furthermore, tubular injury and cast formation observed in controls was minimized by glycine (pathology score, 3.2 +/- 0.4 vs. 1.0 +/- 0.4, P < 0.05). Urinary lactate dehydrogenase (LDH) concentration was elevated by ischemia-reperfusion in the control group (260 +/- 22 U/l), but values were significantly lower by about fourfold (60 +/- 30 U/l) in glycine-fed rats. Similarly, free radical production in urine was significantly lower in glycine-treated animals. Importantly, on postischemic day 1, binding of pimonidazole, an in vivo hypoxia marker, was increased in the outer medulla in controls; however, this phenomenon was prevented by glycine. Two weeks later, mild leukocyte infiltration and interstitial fibrosis were still observed in controls, but not in kidneys from glycine-treated rats. In conclusion, these results indicate that administration of glycine indeed reduces mild ischemia-reperfusion injury in the kidney in vivo, in part by decreasing initial damage and preventing chronic hypoxia.