Georgina Hotter

Macrophage involvement in the kidney repair phase after ischaemia/reperfusion injury

Macrophage infiltration is a common feature of the early phase of renal ischaemia/reperfusion injury. Indeed, it is generally regarded as the cause of tissue injury in this phase, although it is also clear that it can lead to tissue repair in other phases. In order to ascertain whether macrophages are directly involved in the repair/late phase, which follows the pro‐inflammatory and injury process of renal ischaemia/reperfusion, we used two different approaches based on macrophage depletion. Firstly, we produced renal ischaemia in mice that were previously treated with clodronate liposome. Secondly, during reperfusion we re‐injected RAW 264.7 to macrophage‐depleted mice 24 h prior to sacrifice. The results showed that regeneration, as evaluated by stathmin and PCNA markers, was macrophage‐dependent: it was blocked when macrophage depletion was provoked and recovered with macrophage re‐injection. The cytokine profile revealed the influence of the inflammatory environment on kidney repair: pro‐inflammatory cytokines (MCP‐1, MIP‐1α) increased during the early stages of reperfusion, coinciding with low regeneration, and the anti‐inflammatory cytokine IL‐10 increased during the longer periods of reperfusion when regeneration was more evident. We conclude that macrophages induce renal regeneration after ischaemia/reperfusion, depending on the inflammatory milieu. Copyright

Protective Effect of Ischemic Preconditioning on Cold Preservation and Reperfusion Injury Associated With Rat Intestinal Transplantation

ObjectiveTo define the protective effect of ischemic preconditioning on cold ischemia and reperfusion injury associated with intestinal transplantation, and the role of nitric oxide in this process. Summary Background DataIschemia/reperfusion injury continues to be a significant obstacle in small bowel transplantation. Preconditioning is a mechanism that protects against this injury. MethodsTo study the capacity of preconditioning to prevent cold ischemia-associated injury and the inflammatory response associated with intestinal transplantation, the authors studied a control group of animals, cold ischemia groups with or without previous preconditioning and with or without previous administration of L-NAME or NONOS, and intestinal transplantation groups with or without previous preconditioning and with or without previous administration of L-NAME or NONOS. ResultsHistologic findings and the release of lactate dehydrogenase into the preservation solution showed that preconditioning protects against cold ischemic preservation-associated injury. Preconditioning also prevented the inflammatory response associated with intestinal transplantation, measured by the above parameters and by neutrophil recruitment in the intestine. Inhibition of nitric oxide eliminates the protective effect. ConclusionsPreconditioning protects the intestinal grafts from cold preservation and reperfusion injury in the rat intestinal transplantation model. Nitric oxide is involved in this protection.

Minimally invasive silicon probe for electrical impedance measurements in small animals

It is commonly accepted that electrical impedance provides relevant information about the physiological condition of living tissues. Currently, impedance measurements are performed with relatively large electrodes not suitable for studies in small animals due to their poor spatial resolution and to the damage that they cause to the tissue. A minimally invasive needle shaped probe for electrical impedance measurements of living tissues is presented in this paper. This micro-probe consists of four square platinum electrodes (300 microm x 300 microm) on a silicon substrate (9 mm x 0.6 mm x 0.5 mm) and has been fabricated by using standard Si microelectronic techniques. The electrodes are not equally spaced in order to optimise the signal strength and the spatial resolution. Characterisation data obtained indicate that these probes provide high spatial resolution (measurement radius <4 mm) with a useful wide frequency band going from 100 Hz to 100 kHz. A series of in vivo experiments in rat kidneys subjected to ischemia was performed to demonstrate the feasibility of the probes and the measurement system. The impedance modulus and phase were measured at 1 kHz since this frequency is sufficiently low to permit the study of the extracellular medium. The extracellular pH and K+ were also simultaneously measured by using commercial miniaturised Ion Selective Electrodes. The induced ischemia period (45 min) resulted in significant changes of all measured parameters (Delta/Z/ approximately 65%; DeltapH approximately 0.8; DeltaK+ approximately 30 mM).

Modification of oxidative stress in response to intestinal preconditioning

Previous studies have demonstrated that intestinal preconditioning protects the organ from ischemia reperfusion damage. Xanthine oxidase mediating free radical generation contributes to the development of injury associated to ischemia reperfusion. Thus, any process able to modulate the oxygen free radical generation system could attenuate the injury. Also, it is known that nitric oxide is implicated in the preconditioning response. The aim of this work is to determine: (1) the effect of intestinal preconditioning on the xanthine oxidase system, (2) the relevance of this system in the development of injury, and (3) its relationship with nitric oxide. For this purpose, we have determined the activity of the xanthine dehydrogenase/xanthine oxidase system, the levels of its substrate (xanthine), and end-product (uric acid) and oxidant stress status in rat small intestine subjected to ischemic pre-conditioning. The effects of nitric oxide inhibition have also been evaluated. Results show that the percentage of xanthine dehydrogenase to xanthine oxidase conversion, xanthine, uric acid concentration, lipoperoxides, and reduced glutathione were significantly reduced in preconditioned rats irrespectively of nitric oxide inhibition. In summary, this work shows that oxidative stress in intestinal preconditioning is reduced as consequence of the diminished conversion of xanthine dehydrogenase to xanthine oxidase, and also as a consequence of the reduced availability of xanthine.

Infusion of IL-10–expressing cells protects against renal ischemia through induction of lipocalin-2

Ischemia/reperfusion injury is a leading cause of acute renal failure triggering an inflammatory response associated with infiltrating macrophages, which determine disease outcome. To repair the inflammation we designed a procedure whereby macrophages that overexpress the anti-inflammatory agent interleukin (IL)-10 were adoptively transferred. These bone marrow-derived macrophages were able to increase their intracellular iron pool that, in turn, augmented the expression of lipocalin-2 and its receptors. Infusion of these macrophages into rats after 1 h of reperfusion resulted in localization of the cells to injured kidney tissue, caused increases in regenerative markers, and a notable reduction in both blood urea nitrogen and creatinine. Furthermore, IL-10 therapy decreased the local inflammatory profile and upregulated the expression of pro-regenerative lipocalin-2 and its receptors. IL-10-mediated protection and subsequent renal repair were dependent on the presence of iron and lipocalin-2, since the administration of a neutralizing antibody for lipocalin-2 or administration of IL-10 macrophages pretreated with the iron chelating agent deferoxamine abrogated IL-10-mediated protective effects. Thus, adoptive transfer of IL-10 macrophages to ischemic kidneys blunts acute kidney injury. These effects are mediated through the action of intracellular iron to induce lipocalin-2.

Bioimpedance dispersion width as a parameter to monitor living tissues

In the case of living tissues, the spectral width of the electrical bioimpedance dispersions (closely related with the alpha parameter in the Cole equation) evolves during the ischemic periods. This parameter is often ignored in favor of other bioimpedance parameters such as the central frequency or the resistivity at low frequencies. The object of this paper is to analyze the significance of this parameter through computer simulations (in the alpha and beta dispersion regions) and to demonstrate its practical importance through experimental studies performed in rat kidneys during cold preservation. The simulations indicate that the dispersion width could be determined by the morphology of the extra-cellular spaces. The experimental studies show that it is a unique parameter able to detect certain conditions such as a warm ischemia period prior to cold preservation or the effect of a drug (Swinholide A) able to disrupt the cytoskeleton. The main conclusion is that, thanks to the alpha parameter in the Cole equation, the bioimpedance is not only useful to monitor the intra/extra-cellular volume imbalances or the inter-cellular junctions resistance but also to detect tissue structural alterations.

Free radical enhancement promotes leucocyte recruitment through a PAF and LTB4 dependent mechanism

In the present investigation we studied the concerted role of superoxide anion, platelet activating factor (PAF) and leukotriene B4 (LTB4) in the mechanism that results in polymorphonuclear leucocyte accumulation induced by oxygen free radicals in rat pancreas. This was done by comparing the effects of a PAF antagonist (BN-52021), a LTB4 inhibitor (MK-886) and superoxide dismutase (SOD) in a experimental rat model of inflammation elicited by the oxygen free radicals induced via infusion of xanthine/xanthine oxidase. Also, the effect of independent LTB4 infusion has been studied. The results show that increases in polymorphonuclear cell infiltration (evaluated by tissue histology), myeloperoxidase and LTB4 levels induced in pancreas by infusion of xanthine/xanthine oxidase were abolished by the administration of either the PAF antagonist, the LTB4 inhibitor, or SOD. The fact that BN-52021 could prevent neutrophil recruitment and LTB4 synthesis suggests that PAF is a necessary step for subsequent LTB4 synthesis and polymorphonuclear leucocyte accumulation.

Lipocalin-2-induced renal regeneration depends on cytokines

This study investigated whether the renal regeneration occurring in the recovery phase of kidney ischemia-reperfusion (I/R) is mediated by endogenously generated lipocalin-2 (Lcn2). A second objective was to examine whether Lcn2-mediated cell effects could be regulated by the inflammatory cytokines in the environment through their action on Lcn2 receptors (Lcn2R and megalin). Male Swiss mice were subjected to 30 min of renal ischemia with a reperfusion period of 24 h (early reperfusion, expected time for maximum inflammation) and 96 h (late reperfusion, expected time for maximum regeneration). Different experimental groups underwent I/R, I/R with iv anti-mouse Lcn2 monoclonal antibody injected during the early/inflammatory or late/recovery phase, and I/R with proinflammatory cytokine cocktail administration (recombinant mouse IL-1beta, TNF-alpha, and IFN-gamma). Compared with control nonischemic mice, the expression of three proliferation markers (stathmin, PCNA, and Ki-67, analyzed by quantitative RT-PCR) increased significantly in the I/R-treated animals. Blockade of Lcn2 by addition of anti-Lcn2 antibody significantly decreased the expression of these three proliferation markers when administered in the late/reparative phase, but had the opposite effect when administered in the early/inflammatory phase. Proinflammatory cytokine cocktail administration reduced the proliferative effects of Lcn2, and repressed Lcn2R and megalin expression. In conclusion, endogenously generated Lcn2 induces renal cell regeneration depending on the inflammatory cytokines in kidney I/R.

Cisplatin upregulates mitochondrial nitric oxide synthase and peroxynitrite formation to promote renal injury

The mitochondria are a critical target for cisplatin-associated nephrotoxicity. Though nitric oxide formation has been implicated in the toxicity of cisplatin, this formation has not so far been related to a possible activation of mitochondrial nitric oxide synthase (mNOS). We show here that the upregulation of oxide mNOS and peroxynitrite formation in cisplatin treatment are key events that influence the development of the harmful parameters described in cisplatin-associated kidney failure. We confirm this by isolating the mitochondrial fraction of the kidney and across different access routes such as the use of a specific inhibitor of neuronal NOS, L-NPA, a peroxynitrite scavenger, FeTMPyP, and a peroxynitrite donor, SIN-1. The in vitro studies corroborated the information obtained in the in vivo experiments. The administration of cisplatin reveals a clear upregulation in the transcription of neuronal NOS and an increase in the levels of nitrites in the mitochondrial fractions of the kidneys. The upregulated transcription directly affects the cytoskeleton structure and the apoptosis. The inhibition of neuronal NOS reduces the levels of nitrites, cell death, and cytoskeleton derangement. Peroxynitrite is involved in the mechanism promoting the NOS transcription. In addition, in controls SIN-1 imitates the effects of cisplatin. In summary, we demonstrate that upregulation of mNOS in cisplatin treatment is a key component in both the initiation and the spread of cisplatin-associated damage in the kidney. Furthermore, peroxynitrite formation is directly involved in this process.

Tissular prostanoid release, phospholipase A2 activity, and lipid peroxidation in pancreas transplantation

Free radical species have been implicated as important agents in ischemia-reperfusion injury associated to transplantation procedures. This study was carried out to investigate the possible relationship between phospholipase A2 activity (PLA2), lipoperoxidation, and the changes in arachidonic acid metabolism during ischemia reperfusion injury in pancreas transplantation, as well as the effect of a free radical scavenger such as super-oxide dismutase on these changes. For this purpose male Lewis rat groups (n = 7) were classified as follows: group I—control; group II—syngenic pancreas transplantation after 15 min preservation in Collins solution at 4 °C; group III—syngenic pancreas transplantation after 18 hr preservation in the same conditions; group IV—same as III but with administration of SOD (i.v.) immediately before revascularization in the recipient rat. The results indicate that significant increases in PLA2 activity and lipoperoxide levels occur concomitantly with an increase of thromboxane B2 (TXB2) and 6-keto prostaglandin F1α (6-keto PGF1α) in pancreatic tissue after pancreas transplantation. The counteracting effect of a free radical scavenger such as SOD supports the role of oxygen free radicals (OFR) mediating activation of PLA2 and subsequent formation of eicosanoids in pancreas transplantation.