Immaculada Herrero

The enhancement of endogenous cAMP with pituitary adenylate cyclase-activating polypeptide protects rat kidney against ischemia through the modulation of inflammatory response.

BACKGROUND Cyclic nucleotide analogue administration improves ischemia-reperfusion damage in several organs. The neuropeptide pituitary adenylate cyclase-activating polypeptide, PACAP-38, is a potent stimulus to enhance cellular cAMP levels. This study tested the protective effect of enhancing endogenous cAMP levels by PACAP-38 in a model of warm renal ischemia. METHODS Sprague-Dawley rats underwent 40 min of bilateral warm renal ischemia. PACAP-38 continuous infusion began either before ischemia or at 6 hr or 18 hr after ischemia. A mini-osmotic pump infused PACAP-38 throughout 7 days of follow-up. Groups were constructed with sham, ischemic control, and dibutyryl cAMP treated animals, and four PACAP-38 treatment groups, using 16 pmol/hr or 160 pmol/hr of the compound, or delaying its administration by 6 hr or 18 hr after ischemia. Renal function was assessed by means of serum creatinine levels on days 1, 2, 3, and 7 after ischemia. Conventional histology was performed on day 7. Renal myeloperoxidase (MPO) activity, infiltrating CD45+ cells, plasma and tissue cAMP, and serum IL-6 were measured. RESULTS Continuous administration of the high concentration of PACAP-38 ameliorated renal function and morphologic abnormalities induced by warm ischemia. Treatment with dibutyryl cAMP produced morphologic protection but only partial functional effect on the ischemic kidney. A 6-hour delay in the administration of the compound after ischemia offered similar protective effect, whereas an 18-hr delay did not. The neuropeptide clearly increased circulating cAMP after ischemia but not cAMP in renal tissue. PACAP-38 increased circulating IL-6, and minimized renal inflammatory cell infiltration induced by ischemia-reperfusion injury, as evidenced by a reduction of MPO activity and the number of CD45+ cells in ischemic renal tissue. CONCLUSIONS Enhancement of endogenous circulating cAMP with PACAP-38 modulates postischemic inflammatory response and strongly protects from ischemic acute renal failure, even when administration is delayed for 6 hr after injury.

Effect of a platelet-activating factor (PAF) receptor antagonist on hyperacute xenograft rejection; evaluation in a pig kidney-human blood xenoperfusion model

In pig to human discordant xenotransplantation, PAF may contribute to the pathogenesis of hyperacute xenograft rejection (HXR). We examined the release of PAF and the effect of a PAF receptor antagonist (BN 52021) on HXR in a pig kidney–human blood xenoperfusion model. Pig kidneys were perfused with porcine blood (AUTO group, n = 5), human blood (HETER group, n = 6) or human blood plus BN 52021 (BN group, n = 4), respectively. In contrast to HETER kidneys that never produced urine and were rejected in 15–30 min, the administration of BN 52021 induced a partial recovery of glomerular filtration rate and allowed kidneys to function until the end of the study. The release of PAF and soluble P‐selectin, as well as endothelial P‐selectin expression and tissue myeloperoxidase (MPO), were much higher in the HETER than in the AUTO group. HETER and BN kidneys displayed similar natural xenoantibody titres, CH50, PAF, soluble P‐selectin as well as renal immunoglobulin (IgM, IgG, IgA) and complement (C3, C1q) deposition. However, HETER kidneys displayed a full histologic picture of HXR (mainly interstitial haemorrhage and vascular microthrombi) and BN kidneys had only endothelial cell swelling. Also, BN 52021 administration attenuated glomerular and vascular P‐selectin expression and renal tissue MPO activity. We conclude that in the pig kidney–human blood xenoperfusion model, PAF is produced in higher amounts than in the pig kidney–pig blood autologous combination. The administration of BN 52021 exerts a protective effect by means of attenuating the acute inflammatory response and blocking vascular microthrombi formation.

Renal protective effect of liposomed superoxide dismutase in an experimental warm ischemia model

Abstract Superoxide dismutdse (SOD) is a potent scavenger of superoxide radicals produced during normothermic ischemia‐reperfusion. Since it has a short half‐life, its optimal effect is achieved when it is given prior to reperfusion. The inclusion of SOD in liposomes (lipo‐OD) prolongs its half‐life (free SOD: 6 min; lipo‐OD: 4 h). The protective effect of lipo‐OD in a 60‐min bilateral renal warm ischemia model was studied. We divided 60 male Wistar rats between two control groups and five study groups according to the drug used (SOD or lipo‐OD) and to the time of SOD administration (prior to ischemia or prior tb reperfusion). SOD and lipo‐OD were both given at 20 mg/kg endovenously. Weight, diuresis, creatinine per 100 g (Cr/100 g), and creatinine clearance per 100 g (CrC1/100 g) were studied. Conventional renal histology was performed after reperfusion and on day 7. Renal malondialdehyde, 6 keto PGF 1 alpha, and TxB2 tissue levels were studi. ed after reperfusion. Results showed that the renal protective effect of free SOD on warm ischemic‐reperfusion injury depended on the time of administration, being more effective when given before reperfusion. On the other hand, the renal protective effect of liposomed SOD did not depend on the time of administration since efficacy was similar when given before reperfusion or before ischemia. The functional protective effect of liposomed SOD was similar to that of free SOD when they were given prior to reperfusion. Nevertheless, since histological damage observed with liposomed SOD was less than with free SOD, it is suggested that the liposomed galenic form may offer better protection against renal warm ischemia. In addition, liposomed SOD was better at preventing tissue prostanoid generatioh after renal warm ischemic‐reperfusion injury than free SOD. We concluded that liposomed SOD shows a higher renal protective effect against warm ischemia than free SOD.

Changes in renal hemodynamics and physiology after normothermic ischemia in animals supplemented with eicosapentaenoic acid.

Abstract  In order to determine whether treatment of animals with an n‐3 fatty acid, eicosapentaenoic acid (EPA), could modify renal hemodynamics and physiology after normothermic ischemia, we studied 42 Spraque Dawley rats orally supplemented with either olive oil or a purified lysine salt of EPA for 4 weeks. Four experimental groups were established. Three groups were treated with increasing doses of EPA: 20 mg/kg per day (EPA 20), 40 mg/kg per day (EPA 40) and 80 mg/kg per day (EPA 80), and one group was supplemented with iso‐volumetric olive oil (OLI). A control group that received neither EPA nor ischemia was also studied. On day 28, right nephrectomy was performed, followed by 30 min of left renal warm ischemia. Basal arterial pressure and renal blood flow (RBF) were monitored in two kidneys before arterial occlusion and continuously thereafter throughout the experiment in one kidney using an electronic transducer and a flowmeter. From 60 to 120 min after the end of ischemia, urine output (μl/min), glomerular filtration rate (GFR, μl/ min), measured by inulin clearance, and fractional reabsortion of sodium (FRNa) were determined every 20 min. Renal plasma flow (RPF, ml/ min) and renal vascular resistance (VR, mm Hg/ml per min) were calculated. RPF was estimated as RBF (1‐hematocrit). Before ischemia, the mean RPF and RBF were higher in EPA‐fed than in olive oil‐fed animals and after ischemia showed a significantly greater increase in EPA‐fed animals than in olive oil‐fed animals. Mean VR was lower in EPA‐fed animals than in olive oil‐fed animals, both before arterial occlusion and after ischemia. Mean urine output was similar in the OLI and EPA 20 groups, and significantly higher in the EPA 40 and EPA 80 groups than in the control group. GFR was significantly lower in the OLI and EPA 20 groups than in the control group. Finally, the EPA 40 group showed a similar and the EPA 80 group a slightly higher GFR than the control group. We conclude that EPA supplementation provides protection from renal ischemic‐re‐perfusion injury, and this effect is more evident at higher EPA doses.

Functional and pathologic outcome after complement inactivation in a pig kidney-human blood xenoperfusion model

Abstract In pig to human discordant xenotransplantation there is evidence that hyperacute rejection (HAR) is mediated by the binding of natural xenoantibodies (NxAb) to endothelial xenoantigens with subsequent complement system activation. 1 Hence, prolonged graft survival using complement inhibitors such as cobra venom factor, 2 sCR1, 3 or transgenic animals expressing human complement regulatory proteins such as DAF 4 or CD59, 5 has been observed. Thus, complement plays a pivotal role in endothelial barrier disruption and in the development of HAR. 6 Ex vivo organ perfusion is accepted as a useful tool to study HAR in the pig to human discordant combination. 7 Most of the xenoperfusion experiments in this combination have been performed using pig hearts or pig livers; there is less experience in the functional evaluation of pig kidneys perfused with human blood. We designed this work in order to evaluate the functional and pathologic findings in pig kidneys perfused with complement-inactivated human blood.

The ether phospholipids trail: blood timing in renal ischemia-reperfusion injury

THE ABSENCE of blood flow during ischemia causes initial damage, which, if prolonged beyond the reserve limits of each organ, becomes irreversible. Reperfusion allows for return of oxygen delivery to tissues but the reoxygenation itself, necessary for organ recovery, may also increase the injury. This reoxygenation is associated with a massive production of toxic free radical species generated through several cytoplasmatic or mitochondrial mechanisms. Oxidative stress during reperfusion triggers PAF synthesis through the PLA2 pathway, thus inducing polymorphonuclear recruitment. In addition, oxygen free radicals (OFR) may induce the formation of oxidized phospholipids with PAF activity from endothelial cells, as it has been assessed in in vitro studies. Platelet-activating factor (PAF) is a potent bioactive lipid, with a diverse array of biologic effects in isolated systems, but its predominant role is in inflammation. Several studies have suggested the participation of PAF in ischemia and reperfusion (IRI) using PAF antagonists; however, only few of them have measured the release of PAF in the effluents. Measurement of PAF in experimental samples has been done with gas chromatography-mass spectrometry (GC-MS), radioimmunoassays (RIA), or bioassays. Bioassay is the most commonly method to date. All these methods are limited because they measure not only PAF, but also PAF-like activity. In addition there is minimal information about the timing of in vivo generation of PAF after IRI. In the present study we assessed the timing of PAF production after warm renal ischemia in two different animal species.

Effect of human natural xenoantibody depletion and complement inactivation on early pig kidney function.

Preformed xenoreactive natural antibodies (XNA) and complement mediate hyperacute xenograft rejection (HXR) in pig-to-human discordant xenotransplantation. In a pig kidney-human blood xenoperfusion model, we investigated whether XNA depletion and/or human complement inactivation preserved early pig kidney function. Pig kidneys were perfused for 180 min with pig blood (AUTO group, n = 8), human blood (HETER group, n = 6), complement-inactivated human blood (COMi group, n = 5), XNA-depleted human blood (ABd group, n = 5) or complement-inactivated and XNA-depleted human blood (ABd&COMi group, n = 5). HETER kidneys were rejected after 15–30 min and showed vascular microthrombi and interstitial hemorrhages. XNA depletion and/or complement inactivation prevented HXR. The glomerular filtration rate in ABd, COMi and ABd&COMi groups was significantly lower than in the AUTO group. Also, beyond 60 min, the COMi group showed a significantly lower glomerular filtration rate than that observed in ABd and ABd&COMi groups. Kidneys from ABd, COMi and ABd&COMi groups displayed endothelial cell edema, as well as higher soluble P-selectin levels and a higher renal myeloperoxidase content than the AUTO group kidneys. COMi and ABd&COMi groups had a significantly lower renal myeloperoxidase level than the HETER group. Also, in contrast to HETER and ABd groups, these complement-inactivated groups failed to show a positive correlation between P-selectin and renal myeloperoxidase. We also investigated platelet-activating factor (PAF) as possible mediator for these functional and pathologic changes. We found that blood PAF levels were similar in HETER, ABd, COMi and ABd&COMi groups and significantly higher than in the AUTO group. Also, when PAF was added to porcine endothelial cell monolayers, morphological changes due to cytoskeleton contraction were observed, and these changes were prevented by preincubation with a PAF receptor antagonist. In conclusion, although depletion of XNA and/or complement inactivation prevent HXR, the pig kidney function is not preserved at the level of the autologous combination. The PAF overproduction observed in the xenogenic combination, which is independent of the presence of XNA and complement, may be, at least in part, responsible for early endothelial cell morphological changes still present when HXR is prevented.

Effect of the platelet-activating factor antagonist BN-52021 on liver preservation (4°): experimental study in isolated reperfused rat liver model ☆

PRESERVATION INJURY is a major contributor to primary allograft dysfunction and failure after liver transplantation. Several studies have shown that cold ischemia (CI) can damage the sinusoidal liver cells. During this process many inflamatory mediators are released locally and to the blood stream. The damaged endothelial cells produce the activation and adhesion of leukocytes. Finally, the infiltration of polymorphonuclear cells increases the cold ischemic lesion. Platelet-activating factor (PAF) is an inflammatory mediator produced by many cell types. It has been implicated in the microcirculatory failure after cold and warm ischemia. PAF antagonist BN-52021 is a natural compound extracted from ginkgo-biloba tree. Its beneficial effect has been reported in ischemia reperfusion injury of many organs. The objective of our study was to demonstrate that the PAF antagonist BN-52021 can attenuate the preservation injury. The isolated perfused rat liver model was employed for reperfusion evaluation.