Protective effect of milk fat globule-epidermal growth factor-factor VIII after renal ischemia-reperfusion injury in mice.
ABSTRACT: Renal ischemia-reperfusion injury causes acute renal failure, and the hallmarks of renal ischemia-reperfusion injury are inflammation, apoptosis, necrosis, and capillary dysfunction. Milk fat globule-epidermal growth factor-factor VIII (MFG-E8), a membrane-associated secretory glycoprotein, is produced by immune cells and reported to participate in multiple physiologic processes associated with tissue remodeling. We have recently shown that MFG-E8 treatment attenuates organ injury, inflammatory responses, and survival after sepsis through the enhancement of phagocytosis of apoptotic cells. The purpose of this study was to determine whether administration of MFG-E8 attenuates renal ischemia-reperfusion injury.Prospective, controlled, and randomized animal study.: A research institute laboratory.Male C57BL/6J mice (20-25 g).: Renal ischemia-reperfusion injury with bilateral renal pedicle clamping for 45 mins, followed by reperfusion. A recombinant murine MFG-E8 (0.4 ?g/20 g) was given intraperitoneally at the beginning of reperfusion.MFG-E8 levels, organ injury variables, inflammatory responses, histology, apoptosis, and capillary functions were assessed at 1.5 and 20 hrs after reperfusion. A 60-hr survival study was conducted in MFG-E8 and recombinant murine MFG-E8-treated wild-type mice. After renal ischemia-reperfusion injury, MFG-E8 mRNA and protein expressions were significantly decreased in the kidneys and spleen. Treatment with recombinant murine MFG-E8 recovered renal dysfunction, significantly suppressed inflammatory responses, apoptosis, necrosis, and improved capillary functions in the kidneys. In the survival study, MFG-E8 mice showed a significant deterioration and, in contrast, recombinant murine MFG-E8-treated wild-type mice showed a significant improvement of survival compared with vehicle-treated wild-type mice.MFG-E8 can be developed as novel treatment for renal ischemia-reperfusion injury. This protective effect appears to be mediated through the enhancement of apoptotic cell clearance and improvement of capillary functions in the kidneys.
Project description:Hepatic ischemia-reperfusion (I/R) injury is a serious clinical complication that may compromise liver function because of extensive hepatocyte loss. Therefore, the development of novel and effective therapies for hepatic I/R is critical for the improvement of patient outcome. It has been previously shown that administration of milk fat globule-EGF factor VIII (MFG-E8), a membrane-associated secretory glycoprotein, exerts significant beneficial effects under acute inflammatory conditions through multiple physiological processes associated with tissue remodeling.To determine whether administration of recombinant human (rh) MFG-E8 attenuates liver injury in an animal model of hepatic I/R, male adult rats were subjected to 70% hepatic ischemia for 90 min, followed by reperfusion. At the beginning of reperfusion, rats were treated intravenously with normal saline (vehicle) or rhMFG-E8 (160 ?g/kg) over a period of 30 min. MFG-E8 levels and various measurements were assessed 4 h after reperfusion. In addition, survival study was conducted in MFG-E8(-/-) and rhMFG-E8-treated wild-type (WT) mice using a total hepatic ischemia model.Liver and plasma MFG-E8 protein levels were significantly decreased after hepatic I/R. Administration of rhMFG-E8 significantly improved liver injury, suppressed apoptosis, attenuated inflammation and oxidative stress, and downregulated NF-?B pathway. We also noticed that rhMFG-E8 treatment restored the downregulated PPAR-? expression after hepatic I/R. MFG-E8(-/-) mice showed deterioration on survival and, in contrast, rhMFG-E8-treated WT mice showed a significant improvement of survival compared with vehicle-treated WT mice.MFG-E8-mediated multiple physiological events may represent an effective therapeutic option in tissue injury following an episode of hepatic I/R.
Project description:Decreased renal blood flow following an ischemic insult contributes to a reduction in glomerular filtration. However, little is known about the underlying cellular or subcellular mechanisms mediating reduced renal blood flow in human ischemic acute kidney injury (AKI) or acute renal failure (ARF). To examine renal vascular injury following ischemia, intraoperative graft biopsies were performed after reperfusion in 21 cadaveric renal allografts. Confocal fluorescence microscopy was utilized to examine vascular smooth muscle and endothelial cell integrity as well as peritubular interstitial pericytes in the biopsies. The reperfused, transplanted kidneys exhibited postischemic injury to the renal vasculature, as demonstrated by disorganization/disarray of the actin cytoskeleton in vascular smooth muscle cells and disappearance of von Willebrand factor from vascular endothelial cells. Damage to peritubular capillary endothelial cells was more severe in subjects destined to have sustained ARF than in those with rapid recovery of their graft function. In addition, peritubular pericytes/myofibroblasts were more pronounced in recipients destined to recover than those with sustained ARF. Taken together, these data suggest damage to the renal vasculature occurs after ischemia-reperfusion in human kidneys. Preservation of peritubular capillary endothelial integrity and increasing pericytes may be critical to recovery from postischemic AKI.
Project description:Milk fat globule-EGF factor 8 (MFG-E8)/lactadherin participates in several cell surface-mediated regulatory events. Although its mRNA is present in the gut, the physiological roles of MFG-E8 in the intestinal mucosa have not been explored. Here we show that MFG-E8 was expressed in intestinal lamina propria macrophages from mice. Using a wound-healing assay, MFG-E8 was shown to promote the migration of intestinal epithelial cells through a PKCepsilon-dependent mechanism. MFG-E8 bound to phosphatidylserine and triggered reorientation of the actin cytoskeleton in intestinal epithelial cells at the wound edge. Depleting MFG-E8 in mice by administration of anti-MFG-E8 antibody or targeted deletion of the MFG-E8 gene resulted in a slowing of enterocyte migration along the crypt-villus axis and focal mucosal injury. Moreover, in septic mice, intestinal MFG-E8 expression was downregulated, which correlated with intestinal injury, interrupted enterocyte migration, and impaired restitution. Treatment with recombinant MFG-E8 restored enterocyte migration, whereas deletion of MFG-E8 impeded mucosal healing in mice with sepsis. These results suggest that a decrease in intestinal MFG-E8 impairs intestinal mucosal repair in sepsis. Together, our data indicate that MFG-E8 plays an important role in the maintenance of intestinal epithelial homeostasis and the promotion of mucosal healing and suggest that recombinant MFG-E8 may be beneficial for the treatment of bowel injuries.
Project description:Our research group recently demonstrated that pericytes are major sources of the secreted glycoprotein and integrin ligand lactadherin (MFG-E8) in B16 melanoma tumors, and that MFG-E8 promotes angiogenesis via enhanced PDGF-PDGFR? signaling mediated by integrin-growth factor receptor crosstalk. However, sources of MFG-E8 and its possible roles in skin physiology are not well characterized. The objective of this study was to characterize the involvement of MFG-E8 in skin wound healing. In the dermis of normal murine and human skin, accumulations of MFG-E8 were found around CD31(+) blood vessels, and MFG-E8 colocalized with PDGFR?(+), ?SMA(+), and NG2(+) pericytes. MFG-E8 protein and mRNA levels were elevated in the dermis during full-thickness wound healing in mice. MFG-E8 was diffusely present in granulation tissue and was localized around blood vessels. Wound healing was delayed in MFG-E8 knockout mice, compared with the wild type, and myofibroblast and vessel numbers in wound areas were significantly reduced in knockout mice. Inhibition of MFG-E8 production with siRNA attenuated the formation of capillary-like structures in vitro. Expression of MFG-E8 in fibrous human granulation tissue with scant blood vessels was less than that in granulation tissue with many blood vessels. These findings suggest that MFG-E8 promotes cutaneous wound healing by enhancing angiogenesis.
Project description:Milk fat globule-epidermal growth factor factor 8 (MFG-E8) regulates innate immune function by modulating cellular signaling, which is less understood. Herein, we aimed to investigate the direct anti-inflammatory role of MFG-E8 in macrophages by pre-treatment with recombinant murine MFG-E8 (rmMFG-E8) followed by stimulation with LPS in RAW264.7 cells and in peritoneal macrophages, isolated from wild-type (WT) or MFG-E8(-/-) mice. RAW264.7 cells and mouse peritoneal macrophages treated with rmMFG-E8 significantly downregulated LPS-induced TNF-α mRNA by 25% and 24%, and protein levels by 29% and 23%, respectively (P<0.05). Conversely, peritoneal macrophages isolated from MFG-E8(-/-) mice produced 28% higher levels of TNF-α, as compared to WT mice when treated with LPS. In in vivo, endotoxemia induced by intraperitoneal injection of LPS (5 mg/kg BW), at 4 h after induction, serum level of TNF-α was significantly higher in MFG-E8(-/-) mice (837 pg/mL) than that of WT (570 pg/mL, P<0.05). To elucidate the direct anti-inflammatory effect of MFG-E8, we examined STAT3 and its target gene, SOCS3. Treatment with rmMGF-E8 significantly induced pSTAT3 and SOCS3 in macrophages. Similar results were observed in in vivo treatment of rmMFG-E8 in peritoneal cells and splenic tissues. Pre-treatment with rmMFG-E8 significantly reduced LPS-induced NF-κB p65 contents. These data clearly indicated that rmMFG-E8 upregulated SOCS3 which in turn interacted with NF-κB p65, facilitating negative regulation of TLR4 signaling for LPS-induced TNF-α production. Our findings strongly suggest that MFG-E8 is a direct anti-inflammatory molecule, and that it could be developed as a therapy in attenuating inflammation and tissue injury.
Project description:Ischemia reperfusion injury is one of the major complications responsible for delayed graft function in kidney transplantation. Applications to reduce reperfusion injury are essential due to the widespread use of kidneys from deceased organ donors where the risk for delayed graft function is especially prominent. We have recently shown that coating of inflamed or damaged endothelial cells with a unique heparin conjugate reduces thrombosis and leukocyte recruitment. In this study we evaluated the binding capacity of the heparin conjugate to cultured human endothelial cells, to kidneys from brain-dead porcine donors, and to murine kidneys during static cold storage. The heparin conjugate was able to stably bind cultured endothelial cells with high avidity, and to the renal vasculature of explanted kidneys from pigs and mice. Treatment of murine kidneys prior to transplantation reduced platelet deposition and leukocyte infiltration 24?hours post-transplantation, and significantly improved graft function. The present study thus shows the benefits of enhanced protection of the renal vasculature during cold storage, whereby increasing the antithrombotic and anti-adhesive properties of the vascular endothelium yields improved renal function early after transplantation.
Project description:BACKGROUND:Acute kidney injury (AKI) is most commonly caused by sepsis in critically ill patients, and it is associated with high morbidity and mortality. The pathophysiology of sepsis-induced AKI is generally accepted to include direct inflammatory injury, endothelial cell dysfunction, and apoptosis. Milk fat globule-epidermal growth factor-factor VIII (MFG-E8) is a secretory glycoprotein with a known role in the enhancement of apoptotic cell clearance and regulation of inflammation. We hypothesize that administration of recombinant mouse MFG-E8 (rmMFG-E8) can protect mice from kidney injuries caused by sepsis. METHODS:Sepsis was induced in 8-wk-old male C57BL/6 mice by cecal ligation and puncture (CLP). rmMFG-E8 or phosphate-buffered saline (vehicle) was injected intravenously at a dosage of 20 ?g/kg body weight at time of CLP (n = 5-8 mice per group). After 20 h, serum and renal tissue were harvested for various analyses. The renal injury markers blood urea nitrogen (BUN) and creatinine were determined by enzymatic and chemical reactions, respectively. The gene expression analysis was carried out by real-time quantitative polymerase chain reaction. RESULTS:At 20 h after CLP, serum levels of BUN and creatinine were both significantly increased in the vehicle group compared with the sham group, whereas the mice treated with rmMFG-E8 had a significant reduction in BUN and creatinine levels by 28% and 24.1%, respectively (BUN: 197.7 ± 23.6 versus 142.3 ± 20.7 mg/dL; creatinine: 0.83 ± 0.12 versus 0.63 ± 0.06 mg/dL; P < 0.05). Expressions of novel biomarkers of renal tissue injury neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 were also significantly downregulated by 58.2% and 95%, respectively, after treatment with rmMFG-E8. Proinflammatory cytokine interleukin-6 and tumor necrosis factor-? messenger RNA (mRNA) were significantly reduced by 50.8% and 50.3%, respectively, in rmMFG-E8-treated mice compared with vehicle-treated mice. The mRNA levels of the chemokines keratinocyte chemoattractant and macrophage inhibitory protein-2 were reduced by 85.1% and 78%, respectively, in mice treated with rmMFG-E8 compared with the vehicle mice. In addition, the expression of intercellular cell adhesion molecule-1 and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) mRNA was downregulated by 35.6% and 77.8%, respectively, in rmMFG-E8-treated mice compared with the vehicle animals (P < 0.05). CONCLUSIONS:Treatment with rmMFG-E8 reduces renal tissue injury induced by sepsis through inhibiting the production of proinflammatory cytokines and chemokine, as well as through the activation of endothelial cells. Thus, MFG-E8 may have a therapeutic potential for treating AKI induced by sepsis.
Project description:Ischemia-reperfusion injury is an important contributor to acute kidney injury and a major factor affecting early functional recovery after kidney transplantation. We conducted this experiment to investigate the protective effect of induced multipotent stem cell transplantation on renal ischemia-reperfusion injury. Forty rabbits were divided into four groups of 10 rabbits each. Thirty rabbits were used to establish the renal ischemia-reperfusion injury model, and ten rabbits served as the model group and were not treated. Among the 30 rabbits with renal ischemia-reperfusion injury, 10 rabbits were treated with induced peripheral blood mononuclear cells (PBMCs), and 10 other rabbits were treated with noninduced PBMCs. After three weekly treatments, the serum creatinine levels, urea nitrogen levels and urine protein concentrations were quantified. The kidneys were stained with hematoxylin-eosin (HE), periodic acid-Schiff (PAS) and Masson's trichrome and then sent for commercial metabolomic testing. The kidneys of the rabbits in the model group showed different degrees of pathological changes, and the recovery of renal function was observed in the group treated with induced cells. The results indicate that PBMCs differentiate into multipotent stem cells after induction and exert a therapeutic effect on renal ischemia-reperfusion injury.
Project description:Acute kidney injury (AKI) complicated by acute lung injury has a detrimental effect on mortality among critically ill patients. Recently, a renal ischemia-reperfusion (IR) model suggested the involvement of histones and neutrophil extracellular traps (NETs) in the development of distant lung injury after renal IR. Given that recombinant thrombomodulin (rTM) has anti-inflammatory roles by binding to circulating histones, we aimed to clarify its effect on distant lung injury induced by AKI in a murine bilateral renal IR model. Both pretreatment and delayed treatment with rTM significantly decreased pulmonary myeloperoxidase activity, but they did not affect renal dysfunction at 24?h after renal IR. Additionally, rTM mitigated the renal IR-augmented expression of proinflammatory cytokines (tumor necrosis factor-?, interleukin-6, and keratinocyte-derived chemokine), and vascular leakage, as well as the degree of lung damage. Intense histone accumulation and active NET formation occurred in both the kidneys and the lungs; however, rTM significantly decreased the histone and NET accumulation only in the lungs. Administration of rTM may have protective impact on the lungs after renal IR by blocking histone and NET accumulation in the lungs, although no protection was observed in the kidneys. Treatment with rTM may be an adjuvant strategy to attenuate distant lung injury complicating AKI.
Project description:Excessive neutrophil infiltration to the lungs is a hallmark of acute lung injury (ALI). Milk fat globule epidermal growth factor-factor 8 (MFG-E8) was originally identified for phagocytosis of apoptotic cells. Subsequent studies revealed its diverse cellular functions. However, whether MFG-E8 can regulate neutrophil function to alleviate inflammation is unknown. We therefore aimed to reveal MFG-E8 roles in regulating lung neutrophil infiltration during ALI. To induce ALI, C57BL/6J wild-type (WT) and Mfge8(-/-) mice were intratracheally injected with LPS (5 mg/kg). Lung tissue damage was assessed by histology, and the neutrophils were counted by a hemacytometer. Apoptotic cells in lungs were determined by TUNEL, whereas caspase-3 and myeloperoxidase activities were assessed spectrophotometrically. CXCR2 and G protein-coupled receptor kinase 2 expressions in neutrophils were measured by flow cytometry. Following LPS challenge, Mfge8(-/-) mice exhibited extensive lung damage due to exaggerated infiltration of neutrophils and production of TNF-?, MIP-2, and myeloperoxidase. An increased number of apoptotic cells was trapped into the lungs of Mfge8(-/-) mice compared with WT mice, which may be due to insufficient phagocytosis of apoptotic cells or increased occurrence of apoptosis through the activation of caspase-3. In vitro studies using MIP-2-mediated chemotaxis revealed higher migration of neutrophils of Mfge8(-/-) mice than those of WT mice via increased surface exposures to CXCR2. Administration of recombinant murine MFG-E8 reduces neutrophil migration through upregulation of GRK2 and downregulation of surface CXCR2 expression. Conversely, these effects could be blocked by anti-?(v) integrin Abs. These studies clearly indicate the importance of MFG-E8 in ameliorating neutrophil infiltration and suggest MFG-E8 as a novel therapeutic potential for ALI.