W.A. Buurman

Apoptosis and inflammation in renal reperfusion injury

Ischemia followed by reperfusion (I/R) has cardinal implications in the pathogenesis of organ transplantation and rejection. Apoptosis and inflammation are central mechanisms leading to organ damage in the course of renal I/R. General aspects of apoptosis, morphology, induction, and biochemistry are discussed. Activated caspases, the classical effector enzymes of apoptosis, are able to induce not only apoptosis but also inflammation after I/R in experimental models. This redefines the involvement of apoptosis in I/R injury toward a central and functional role in the development of organ damage. Our purpose is to assess aspects of apoptosis and inflammation in terms of involvement in the pathogenesis of I/R-induced organ damage. Moreover, the implications of recent experimental advances for diagnosis and treatment of renal I/R injury in clinical practice will be discussed.

Biological modulation of renal ischemia-reperfusion injury.

Purpose of reviewBiological modulation of renal ischemia–reperfusion injury holds the potential to reduce the incidence of early graft dysfunction and to safely expand the donor pool with kidneys that have suffered prolonged ischemic injury before organ recovery. Recent findingsIn the current review, we will discuss clinical studies that compare kidney transplant recipients with and without early graft dysfunction in order to elucidate the pathophysiology of ischemic acute allograft injury. We will specifically review the mechanisms leading to depression of the glomerular filtration rate and activation of the innate immune system in response to tissue injury. SummaryWe conclude that the pathophysiology of delayed graft function after kidney transplantation is complex and shares broad similarity with rodent models of ischemic acute kidney injury. Given the lack of specific therapies to prevent delayed graft function in transplant recipients, comprehensive efforts should be initiated to translate the promising findings obtained in small animal models into clinical interventions that attenuate ischemic acute kidney injury after transplantation.

Reduction of circulating redox-active iron by apotransferrin protects against renal ischemia-reperfusion injury

Background. Warm ischemia-reperfusion (I/R) injury plays an important role in posttransplant organ failure. In particular, organs from marginal donors suffer I/R injury. Although iron has been implicated in the pathophysiology of renal I/R injury, the mechanism of iron-mediated injury remains to be established. The authors therefore investigated the role of circulating redox-active iron in an experimental model for renal I/R injury. Methods. Male Swiss mice were subjected to unilateral renal ischemia for 45 min, followed by contralateral nephrectomy and reperfusion. To investigate the role of circulating iron, mice were treated with apotransferrin, an endogenous iron-binding protein, or iron-saturated apotransferrin (holotransferrin). Results. Renal ischemia induced a significant increase in circulating redox-active iron levels during reperfusion. Apotransferrin, in contrast to holotransferrin, reduced the amount of circulating redox-active iron and abrogated renal superoxide formation. Apotransferrin treatment did not affect I/R-induced renal apoptosis, whereas holotransferrin aggravated apoptotic cell death. Apotransferrin, in contrast to holotransferrin, inhibited the influx of neutrophils. Both apo- and holotransferrin reduced I/R-induced complement deposition, indicating that the effects of transferrin are differentially mediated by its iron and protein moiety. Finally, apotransferrin, in contrast to holotransferrin, dose-dependently inhibited the loss of renal function induced by ischemia. Conclusions. Redox-active iron is released into the circulation in the course of renal I/R. Reducing the amount of circulating redox-active iron by treatment with apotransferrin protects against renal I/R injury, inhibiting oxidative stress, inflammation, and loss of function. Apotransferrin could be used in the treatment of acute renal failure, as seen after transplantation of ischemically damaged organs.

Apoptosis and chemokine induction after renal ischemia-reperfusion.

Background. One of the earliest prerequisites for the development of inflammation after ischemia-reperfusion (I/R) is local chemokine expression. We recently demonstrated that apoptosis, characterized by intracellular caspase-activation, contributes to the development of inflammation after I/R. Methods. The contribution of apoptosis was investigated using the pan-caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F in a murine model of renal I/R. Renal expression of the chemokines macrophage inflammatory protein-2 (MIP-2) and KC was studied using RT-PCR and immunohistology. Measuring myeloperoxidase activity and serum ureum and creatinine levels assessed neutrophil influx and kidney dysfunction. Results. We demonstrate renal up-regulation of KC and MIP-2 after 1 to 16 hr of reperfusion. Treatment with the caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F effectively prevented I/R-induced renal apoptosis, KC, and MIP-2 up-regulation after 2 hr of reperfusion as well as neutrophil influx and functional impairment after 24 hr of reperfusion. Conclusions. These data for the first time show that chemokine induction following I/R is dependent on caspase activation.

Urine based detection of intestinal mucosal cell damage in neonates with suspected necrotising enterocolitis

Necrotising enterocolitis (NEC) is a severe gastrointestinal disease with a mortality of 20–40%, affecting predominantly premature neonates.1 In the early phase of the disease NEC continues to present a diagnostic challenge for the attending clinician. The signs and symptoms are often non-specific, including gastrointestinal problems such as abdominal distension and feeding intolerance, which are also among the most prevalent presenting features of neonatal sepsis.2 The diagnosis is further hampered by the limited diagnostic accuracy of the laboratory and radiological tests currently in use.3,4 Histopathologically, NEC is characterised by intestinal coagulative or ischaemic necrosis, starting at the mucosa and extending into the submucosa and muscularis externa.1 We sought a non-invasive test to find evidence of enterocyte cell death in infants with gastrointestinal symptoms suspicious of NEC, in order to differentiate NEC from other neonatal diseases that present with abdominal signs. Intestinal fatty acid binding protein (I-FABP) has been reported to be a useful plasma marker for early enterocyte cell death.5,6 The small (14–15 kDa) cytosolic I-FABP is specifically present in mature enterocytes of small and large intestine and is released as soon as cell membrane integrity is compromised. I-FABP is present in very small amounts in the plasma of healthy individuals, probably representing the normal turnover of enterocytes, but levels …

Lipopolysaccharide-binding protein is vectorially secreted and transported by cultured intestinal epithelial cells and is present in the intestinal mucus of mice.

Lipopolysaccharide-binding protein (LBP) is an important modulator of the host’s response to endotoxin. In a previous study, we found evidence for the synthesis of LBP by intestinal epithelial cells. In this study, we explored the polarity of LBP secretion by these cells. Polarized monolayers of Caco-2 cells were used as intestinal mucosa model. Cells were stimulated apically or basally with cytokines, and LBP secretion was analyzed. Furthermore, the presence of LBP in intestinal mucus of healthy and endotoxemic mice was studied using a mucus-sampling technique. The constitutive unipolar LBP secretion from the apical cell surface was markedly enhanced when cells were exposed to cytokines at their apical surface. However, bioactive LBP was secreted from both cell surfaces after basolateral stimulation of cells. Cytokines also influenced the secretion of the acute phase proteins serum amyloid A, apoA-I, and apoB from both surfaces of Caco-2 cells. Furthermore, transport of exogenous LBP from the basolateral to the apical cell surface was demonstrated. In line with these in vitro data, the presence of LBP in intestinal mucus was strongly enhanced in mice after a challenge with endotoxin. The results indicate that LBP is present at the mucosal surface of the intestine, a phenomenon for which secretion and transport of LBP by intestinal epithelial cells may be responsible.

Histological assessment of preimplantation biopsies may improve selection of kidneys from old donors after cardiac death

Kidneys from old donors after cardiac death (DCD) may increase the donor pool but the prognosis of these kidneys is unsatisfactory. To improve these results, we retrospectively evaluated the diagnostic utility of published selection algorithms for old donor kidneys. We studied all DCD kidney transplantations between January 1, 1994 and July 1, 2005 at our institution (n = 199). Selection algorithms were evaluated in the subset of kidney transplantations from donors aged 60 years or older (n = 52). For histological assessment of kidney biopsies, glomerulosclerosis, tubular atrophy, interstitial fibrosis and vascular narrowing were blindly scored. Functional kidney weight was calculated as renal mass multiplied by the fraction of nonsclerosed glomeruli. Graft function and survival of kidneys from DCD aged 60 years or older were inferior to those from younger DCD. Histological scores were associated with kidney function and graft survival of old DCD kidney transplantations. Functional kidney weight was associated with kidney function but not graft survival, while donor glomerular filtration rate (GFR), donor age and machine perfusion characteristics were associated with neither of the clinical outcomes of interest. We conclude that histological assessment of preimplantation biopsies may improve the selection of kidneys from old DCD and may therefore contribute to expansion of the donor pool.

Additional value of serum I-FABP levels for evaluating celiac disease activity in children

Abstract Objectives. Addition of a non-invasive marker for intestinal damage to the currently used parameters for celiac disease activity (symptoms, serologic tests and biopsy) might further improve clinical management of celiac disease (CD). Intestinal fatty acid binding protein (I-FABP) is a cytosolic enterocyte protein and sensitive marker for enterocyte damage in the small intestine. We investigated whether serum I-FABP levels can reliably identify villous atrophy in children with a positive CD antibody screening. Moreover, the recovery of I-FABP levels after gluten free diet (GFD) was studied. Methods. I-FABP levels were analyzed retrospectively in 49 children with biopsy proven CD and in 19 patients with a positive screening but without histological confirmation of CD. Blood was collected before biopsy and repeatedly after the onset of GFD. Results. Initial I-FABP concentrations in CD (median 458 pg/ml) were significantly (p < 0.001) elevated compared to controls (median 20 pg/ml). In the control group, only two of 19 children were found to have elevated I-FABP levels, of which one was subsequently diagnosed with CD after gluten challenge. I-FABP concentrations correlated with severity of villous atrophy. In all CD patients, I-FABP levels decreased quickly after GFD and normalized in 80% of patients within 12 weeks. Conclusions. Elevated I-FABP levels accurately predict villous atrophy in children with a positive serologic test for CD (positive predictive value 98%). In addition, measurement of I-FABP enables monitoring the response to GFD.

Plasma Intestinal Fatty Acid-Binding Protein Levels Correlate With Morphologic Epithelial Intestinal Damage in a Human Translational Ischemia-reperfusion Model.

Background and Aim: Intestinal fatty acid–binding protein (I-FABP) is a useful marker in the detection of intestinal ischemia. However, more insight into the test characteristics of I-FABP release is needed. This study aimed to investigate the relationship between plasma I-FABP levels and the severity of ischemic mucosal injury, and define the clinical usefulness of systemic I-FABP following ischemia. Methods: In a human experimental model, 6 cm of the jejunum, to be removed for surgical reasons, was selectively exposed to either 15, 30, or 60 minutes of ischemia (I) followed by 30 and 120 minutes of reperfusion (R). Blood and tissue was sampled at all time points. Arteriovenous (V−A) concentration differences of I-FABP were measured. Tissue sections were stained with hematoxylin/eosin, and villus height was measured to score epithelial damage. Results: Histologic analysis showed only minor reversible intestinal damage following 15I and 30I; however, severe irreversible epithelial damage was observed in the jejunum exposed to 60I. I-FABP V−A differences paralleled the degree of tissue damage over time [7.79 (±1.8) ng/mL, 128.6 (±44.2) ng/mL, 463.3 (±139.8) ng/mL for 15I, 30I and 60I, respectively]. A good correlation was found between histologic epithelial damage and V−A I-FABP (r=−0.82, P<0.001). Interestingly, systemic I-FABP levels were significantly increased after 60I of this short small intestinal segment. Conclusions: This study demonstrates the relationship between the duration of ischemia and the extent of tissue damage, which is reflected by I-FABP V−A plasma levels. In addition, systemic I-FABP levels appear valuable in detecting irreversible intestinal ischemia-reperfusion damage.

Loss of enterocyte mass is accompanied by diminished turnover of enterocytes after myeloablative therapy in haematopoietic stem-cell transplant recipients

BACKGROUND Intestinal mucosal barrier injury (MBI), resulting from myeloablative conditioning for haematopoietic stem-cell transplantation (HSCT), is an important cause of morbidity. Despite its frequency, recognition presents a challenge, while the aetiology needs still to be unravelled. The relationship between enterocyte mass and enterocyte loss was explored by examining citrulline serum levels and by assessing circulating intestinal fatty acid-binding protein (I-FABP) and ileal bile acid-binding protein (I-BABP), proteins released by dying mature enterocytes. PATIENTS AND METHODS Thirty-four adult patients with haematological malignancy received allogeneic HSCT (HSCT day 0) 12 days after being given idarubicin, cyclophosphamide and total body irradiation as myeloablative conditioning, a regimen known to induce oral and intestinal MBI. Serum levels of citrulline, I-FABP and I-BABP were measured on HSCT days -12, -6, 0, +7, +14 and +21. RESULTS Myeloablative conditioning resulted in a significant decrease in serum citrulline with the nadir on HSCT day +7; thereafter, levels rose gradually. Simultaneously, a significant decrease in I-FABP and I-BABP levels occurred from the day of transplant until day +14. CONCLUSIONS Simultaneous reduction and subsequent increase of citrulline and I-FABP and I-BABP levels following cytotoxic treatment show that enterocyte mass corresponds to lower rate of dying enterocytes, indicating reduced turnover of enterocytes. Assessment of enterocyte turnover and mass offers opportunities for evaluation of new MBI therapies.