Francesco Staffieri

Therapeutic Targeting of Classical and Lectin Pathways of Complement Protects from Ischemia-Reperfusion-Induced Renal Damage

Ischemia-reperfusion injury is the major cause of delayed graft function in transplanted kidneys, an early event significantly affecting long-term graft function and survival. Several studies in rodents suggest that the alternative pathway of the complement system plays a pivotal role in renal ischemia-reperfusion injury. However, limited information is currently available from humans and larger animals. Here we demonstrated that 30 minutes of ischemia resulted in the induction of C4d/C1q, C4d/MLB, and MBL/MASP-2 deposits in a swine model of ischemia-reperfusion injury. The infusion of C1-inhibitor led to a significant reduction in peritubular capillary and glomerular C4d and C5b-9 deposition. Moreover, complement-inhibiting treatment significantly reduced the numbers of infiltrating CD163(+), SWC3a(+), CD4a(+), and CD8a(+) cells. C1-inhibitor administration led to significant inhibition of tubular damage and tubular epithelial cells apoptosis. Interestingly, we report that focal C4d-deposition colocalizes with C1q and MBL at the peritubular and glomerular capillary levels also in patients with delayed graft function. In conclusion, we demonstrated the activation and a pathogenic role of classical and lectin pathways of complement in a swine model of ischemia-reperfusion-induced renal damage. Therefore, inhibition of these two pathways might represent a novel therapeutic approach in the prevention of delayed graft function in kidney transplant recipients.

Inhomogeneity of lung parenchyma during the open lung strategy: a computed tomography scan study.

RATIONALE The open lung strategy aims at reopening (recruitment) of nonaerated lung areas in patients with acute respiratory distress syndrome, avoiding tidal alveolar hyperinflation in the limited area of normally aerated tissue (baby lung). OBJECTIVES We tested the hypothesis that recruited lung areas do not resume elastic properties of adjacent baby lung. METHODS Twenty-five anesthetized, mechanically ventilated pigs were studied. Four lung-healthy pigs served as controls and the remaining 21 were divided into three groups (n = 7 each) in which lung injury was produced by surfactant lavage, lipopolysaccharide infusion, or hydrochloride inhalation. Computed tomography scans, respiratory mechanics, and gas exchange parameters were recorded under three conditions: at baseline, during lung recruitment maneuver, and at end-expiration and end-inspiration when ventilating after an open lung protocol. MEASUREMENTS AND MAIN RESULTS During recruitment maneuver and open lung protocol, the gas volume entering the insufficiently aerated compartment was 96% (75-117%) and 48% (41-63%) (median [interquartile range]) of the functional residual capacity measured before and at zero end-expiratory pressure, respectively. Nonetheless, the volume of hyperinflated lung increased during both recruitment maneuver (by 1-28% of total lung volume; P < 0.01) and open lung protocol ventilation at end-inspiration (by 1-15% of total lung volume; P < 0.01). Regional elastance of recruited lung tissue was consistently higher than that of the baby lung regardless of the ARDS model (P < 0.01). CONCLUSIONS Alveolar recruitment is not protective against hyperinflation of the baby lung because lung parenchyma is inhomogeneous during ventilation with the open lung strategy.

Endothelial-to-mesenchymal transition and renal fibrosis in ischaemia/reperfusion injury are mediated by complement anaphylatoxins and Akt pathway

BACKGROUND Increasing evidence demonstrates a phenotypic plasticity of endothelial cells (ECs). Endothelial-to-mesenchymal transition (EndMT) contributes to the development of tissue fibrosis. However, the pathogenic factors and signalling pathways regulating this process in ischaemia/reperfusion (I/R) injury are still poorly understood. METHODS We investigated the possible role of complement in the induction of this endothelial dysfunction in a swine model of renal I/R injury by using recombinant C1 inhibitor in vivo. RESULTS Here, we showed that I/R injury reduced the density of renal peritubular capillaries and induced tissue fibrosis with generation of CD31(+)/α-SMA(+) and CD31(+)/FPS-1(+) cells indicating EndMT. When we inhibited complement, the process of EndMT became rare, with preserved density of peritubular capillaries and significant reduction in renal fibrosis. When we activated ECs by anaphylatoxins in vitro, C3a and C5a led to altered endothelial phenotype with increased expression of fibroblast markers and decrease expression of specific endothelial markers. The activation of Akt pathway was pivotal for the C3a and C5a-induced EndMT in vitro. In accordance, inhibition of complement in vivo led to the abrogation of Akt signalling, with hampered EndMT and tissue fibrosis. CONCLUSIONS Our data demonstrate a critical role for complement in the acute induction of EndMT via the Akt pathway. Therapeutic inhibition of these systems may be essential to prevent vascular damage and tissue fibrosis in transplanted kidney.

Physiological effects of an open lung ventilatory strategy titrated on elastance-derived end-inspiratory transpulmonary pressure: Study in a pig model*

Rationale:In the presence of increased chest wall elastance, the airway pressure does not reflect the lung-distending (transpulmonary) pressure. Objective:To compare the physiological effects of a conventional open lung approach titrated for an end-inspiratory airway opening plateau pressure (30 cm H2O) with a transpulmonary open lung approach titrated for a elastance-derived end-inspiratory plateau transpulmonary pressure (26 cm H2O), in a pig model of acute respiratory distress syndrome (HCl inhalation) and reversible chest wall mechanical impairment (chest wall and abdomen restriction). Methods:In eight pigs, physiological parameters and computed tomography were recorded under three conditions: 1) conventional open lung approach, normal chest wall; 2) conventional open lung approach, stiff chest wall; and 3) transpulmonary open lung approach, stiff chest wall. Measurements and Main Results:As compared with the normal chest wall condition, at end-expiration non aerated lung tissue weight was increased by 116 ± 68 % during the conventional open lung approach and by 28 ± 41 % during the transpulmonary open lung approach (p < .01), whereas cardiac output was decreased by 27 ± 19 % and 22 ± 14 %, respectively (p = not significant). Conclusion:In this model, the end-inspiratory transpulmonary open lung approach minimized the impact of chest wall stiffening on alveolar recruitment without causing hemodynamic impairment.

Effects of two fractions of inspired oxygen on lung aeration and gas exchange in cats under inhalant anaesthesia

OBJECTIVE To compare the effects of two fractions of inspired oxygen (FiO(2)) (0.4 and 1) on lung aeration and gas exchange during general anaesthesia in cats. STUDY DESIGN Randomized, blinded, controlled study. ANIMALS Thirty healthy, mixed breed, client owned female cats. MATERIALS AND METHODS Cats were premedicated intramuscularly with acepromazine (0.03 mg kg(-1)) and medetomidine (0.015 mg kg(-1)). Anaesthesia was induced with propofol (5 mg kg(-1)) and, after orotracheal intubation, maintained with isoflurane carried by either 100% oxygen (G100, n=15) or an oxygen-air mixture with 40% oxygen (G40, n=15). All cats were placed in dorsal recumbency and breathed spontaneously throughout the entire procedure. Following surgery (ovariectomy), a spiral computed tomography (CT) of the thorax was performed, arterial oxygen (PaO(2)) and carbon dioxide (PaCO(2)) tensions were measured and alveolar-arterial gradient of oxygen [P(A-a)O(2) ] calculated. The CT images were analysed for lung aeration by the analysis of radiograph attenuations (Hounsfield units, HU), according to the following classification: hyperinflated area (-1000 to -900 HU), normally aerated area (-900 to -500 HU), poorly aerated area (-500 to -100 HU) and non-aerated area (-100 to +100 HU). The groups were compared using one-way anova. RESULTS Compared to G100, the normally-aerated lung area was significantly greater and the poorly-aerated and non-aerated areas were significantly smaller in G40. PaCO(2) was similar in both groups. PaO(2) and P(A-a)O(2) were significantly higher in G100. In both groups, pulmonary atelectasis developed preferentially in the caudal lung fields. CONCLUSION In cats anaesthetised with isoflurane, the administration of an FiO(2) of >0.9 significantly impaired lung aeration and gas exchange as compared to an FiO(2) of 0.4. CLINICAL RELEVANCE An FiO(2) of 0.4 may better preserve lung aeration and gas exchange in anaesthetised spontaneously breathing cats but monitoring is essential to ensure oxygenation is adequate.

Effects of positive end-expiratory pressure on anesthesia-induced atelectasis and gas exchange in anesthetized and mechanically ventilated sheep.

OBJECTIVE To evaluate the effects of 10 cm H(2)O of positive end-expiratory pressure (PEEP) on lung aeration and gas exchange in mechanically ventilated sheep during general anesthesia induced and maintained with propofol. ANIMALS 10 healthy adult Bergamasca sheep. PROCEDURES Sheep were sedated with diazepam (0.4 mg/kg, IV). Anesthesia was induced with propofol (5 mg/kg, IV) and maintained with propofol via constant rate infusion (0.4 mg/kg/min). Muscular paralysis was induced by administration of vecuronium (25 microg/kg, bolus IV) to facilitate mechanical ventilation. After intubation, sheep were positioned in right lateral recumbency and mechanically ventilated with pure oxygen and zero end-expiratory pressure (ZEEP). After 60 minutes, 10 cm H(2)O of PEEP was applied for 20 minutes. Spiral computed tomography of the thorax was performed, and data were recorded for hemodynamic and gas exchange variables and indicators of respiratory mechanics after 15 (T(15)), 30 (T(30)), and 60 (T(60)) minutes of ZEEP and after 20 minutes of PEEP (T(PEEP)). Computed tomography images were analyzed to determine the extent of atelectasis before and after PEEP application. RESULTS At T(PEEP), the volume of poorly aerated and atelectatic compartments was significantly smaller than at T(15), T(30), and T(60), which indicated that there was PEEP-induced alveolar recruitment and clearance of anesthesia-induced atelectasis. Arterial oxygenation and static respiratory system compliance were significantly improved by use of PEEP. CONCLUSIONS AND CLINICAL RELEVANCE Pulmonary atelectasis can develop in anesthetized and mechanically ventilated sheep breathing pure oxygen; application of 10 cm H(2)O of PEEP significantly improved lung aeration and gas exchange.

Low respiratory rate plus minimally invasive extracorporeal Co2 removal decreases systemic and pulmonary inflammatory mediators in experimental Acute Respiratory Distress Syndrome.

Objective:The Acute Respiratory Distress Syndrome Network protocol recommends limiting tidal volume and plateau pressure; it also recommends increasing respiratory rate to prevent hypercapnia. We tested a strategy that combines the low tidal volume with lower respiratory rates and minimally invasive CO2 removal. Subjects:Ten lung-damaged pigs (instilled hydrochloride). Interventions:Two conditions randomly applied in a crossover fashion: the Acute Respiratory Distress Syndrome Network protocol and the Acute Respiratory Distress Syndrome Network protocol plus lower respiratory rate plus minimally invasive Co2 removal. A similar arterial Co2 partial pressure was targeted in the two conditions. Measurements and Main Results:Physiological parameters, computed tomography scans, plasma and bronchoalveolar lavage concentrations of interleukin-1&bgr;, interleukin-6, interleukin-8, interleukin-10, interleukin-18, and tumor necrosis factor-&agr;. During the lower respiratory rate condition, respiratory rate was reduced from 30.5 ± 3.8 to 14.2 ± 3.5 (p < 0.01) breaths/min and minute ventilation from 10.4 ± 1.6 to 4.9 ± 1.7 L/min (p < 0.01). The extracorporeal device removed 38.9% ± 6.1% (79.9 ± 18.4 mL/min) of CO2 production. During the lower respiratory rate condition, interleukin-6, interleukin-8, and tumor necrosis factor-&agr; concentrations were significantly lower in plasma; interleukin-6 and tumor necrosis factor-&agr; concentrations were lower in bronchoalveolar lavage, whereas the concentrations of the other cytokines remained unchanged. Conclusion:The strategy of lower respiratory rate plus minimally invasive extracorporeal CO2 removal was feasible and safe and, as compared with the Acute Respiratory Distress Syndrome Network protocol, reduced the concentrations of some, but not all, of the tested cytokines without affecting respiratory mechanics, gas exchange, and hemodynamics.

Use of the oxygen content-based index, Fshunt, as an indicator of pulmonary venous admixture at various inspired oxygen fractions in anesthetized sheep

OBJECTIVE To evaluate the use of the oxygen content-based index, Fshunt, as an indicator of venous admixture (Qs/Qt) at various fractions of inspired oxygen (FIO(2)s) in anesthetized sheep undergoing Flung or 2-lung ventilation. Animals-6 healthy adult female sheep. PROCEDURES Sheep were anesthetized and administered 5 different FIO(2)s (0.21, 0.40, 0.60, 0.80, and 1.00) in random order during 2-lung mechanical ventilation. Arterial and mixed venous blood samples were obtained at each FIO(2) after a 15-minute stabilization period. Vital capacity alveolar recruitment maneuvers were performed after blood collection. The previously used FIO(2) sequence was reversed for sample collection during Flung ventilation. Blood samples were analyzed for arterial, pulmonary end-capillary, and mixed venous oxygen content and partial pressure and for hemoglobin concentration. Oxygen hemoglobin saturation, Qs/Qt, Fshunt, and oxygen tension-based indices (OTIs; including PAO(2):FIO(2), alveolar-arterial difference in partial pressure of oxygen [PAO(2) - PAO(2)], [PAO(2) - Pao(2)]:FIO(2), [PAO(2) - PAO(2)]:PAO(2), and PAO(2):PAO(2)) were calculated at each FIO(2); associations were evaluated with linear regression analysis, concordance, and correlation tests. Intermethod agreement between Qs/Qt and Fshunt was tested via Bland-Altman analysis. RESULTS Strong and significant associations and substantial agreement were detected between Fshunt and Qs/Qt. Relationships between OTIs and Qs/Qt varied, but overall correlations were weak. Conclusions and Clinical Relevance-Whereas OTIs were generally poor indicators of Qs/Qt, Fshunt was a good indicator of Qs/Qt at various FIO(2)s, regardless of the magnitude of Qs/Qt, and could be potentially used as a surrogate for Qs/Qt measurements in healthy sheep.

Complement Modulation of Anti-Aging Factor Klotho in Ischemia/Reperfusion Injury and Delayed Graft Function.

Klotho is an anti‐aging factor mainly produced by renal tubular epithelial cells (TEC) with pleiotropic functions. Klotho is down‐regulated in acute kidney injury in native kidney; however, the modulation of Klotho in kidney transplantation has not been investigated. In a swine model of ischemia/reperfusion injury (IRI), we observed a remarkable reduction of renal Klotho by 24 h from IRI. Complement inhibition by C1‐inhibitor preserved Klotho expression in vivo by abrogating nuclear factor kappa B (NF‐kB) signaling. In accordance, complement anaphylotoxin C5a led to a significant down‐regulation of Klotho in TEC in vitro that was NF‐kB mediated. Analysis of Klotho in kidneys from cadaveric donors demonstrated a significant expression of Klotho in pre‐implantation biopsies; however, patients affected by delayed graft function (DGF) showed a profound down‐regulation of Klotho compared with patients with early graft function. Quantification of serum Klotho after 2 years from transplantation demonstrated significant lower levels in DGF patients. Our data demonstrated that complement might be pivotal in the down‐regulation of Klotho in IRI leading to a permanent deficiency after years from transplantation. Considering the anti‐senescence and anti‐fibrotic effects of Klotho at renal levels, we hypothesize that this acquired deficiency of Klotho might contribute to DGF‐associated chronic allograft dysfunction.

Survival of bone marrow mesenchymal stem cells labelled with red fluorescent protein in an ovine model of collagenase-induced tendinitis

OBJECTIVE The aim of this study was to track the survival and efficacy of allogeneic bone marrow mesenchymal stem cells (BM-MSC) marked with red fluorescent protein (BM-MSCRFP) in an ovine model of collagenase-induced tendinopathy. METHODS Bone marrow was harvested from one donor sheep and BM-MSC were isolated, cultivated and transfected with red fluorescent protein (BM-MSCRFP). Collagenase was injected into both Achilles tendons in the remaining nine sheep. After two weeks the left tendon was injected with a solution of 6 x 10⁶ BM-MSCRFP and fibrin glue, while only fibrin glue was administered to the contra-lateral tendon in each sheep. After three, four and six weeks the tendons were harvested and evaluated for morphology, collagen I deposition, presence of CD34+ cells, and fluorescent labelled BM-MSC. RESULTS We demonstrated that delivery of BM-MSC into tendon lesions had positive effects on the injured tendons. The BM-MSCRFP survived at three, four and six weeks after treatment, leading to better quality healing of tendons as compared to the controls, where no labelled cells were detected. Interestingly, we demonstrated high expression of CD34+ cells in tendons that had been treated with BM-MSCRFP. CLINICAL RELEVANCE Mesenchymal stem cell allografts have a positive effect on tendon healing and local injection of BM-MSC directly into the tendon allows the homing of BM-MSC for good efficiency of engraftment.