Proanthocyanidin Protects Intestine and Remote Organs Against Mesenteric Ischemia/Reperfusion Injury

Background  Intestinal ischemia/reperfusion (IR) induces a systemic inflammatory response and releases harmful substances that may affect the function and integrity of distant organs such as lung, liver, and kidney. We conducted this study to find out if proanthocyanidins (PA) has protective effects against mesenteric IR injury and mesenteric IR-induced intestinal and distant organ injury. Materials and methods  Thirty-two Sprague-Dawley rats were divided into four groups: control, control + PA, IR, IR + PA. The IR and IR + PA groups were subjected to mesenteric arterial ischemia for 60 min and reperfusion for 6 h. The Control + PA and IR + PA groups were administered PA (100 mg/kg/day via oral gavage) for 7 days prior to injury insult. We collected ileal and distant organ tissues, such as pulmonary, hepatic, and kidney specimens to measure tissue levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), and nitrite plus nitrate (NO_x), and we then evaluated histological changes. Results  In the IR group, significant increases in MDA and NO_x levels and significant increases in SOD and GPx activities of intestine, liver, kidney, and lung were observed. The MDA and NO_x levels were significantly lower, as were the SOD and GPx activities in the IR + PA group than that in the IR group. Although the intestine and distant organs damage scores were significantly higher in the IR group, these injuries were prevented by PA in the IR + PA group. Conclusions  This study demonstrates that PA has a significant effect in the protection of the intestine and the remote organs against mesenteric IR injury.     

Parstatin Prevents Renal Injury following Ischemia/Reperfusion and Radiocontrast Administration

Background/Aims: Parstatin is a 41-mer peptide formed by proteolytic cleavage on activation of the protease-activated receptor 1. Parstatin was recently found to be cardioprotective against myocardial ischemia/reperfusion (IR) injury. In the present study, it was hypothesized that parstatin would protect the kidneys in acute renal failure. Methods: We investigated the effects of parstatin on the renal dysfunction and injury caused either by renal IR injury or contrast-induced nephropathy (CIN) in two animal models. Renal IR injury was induced in rats by bilateral occlusion of renal arteries and veins for 45 min followed by 4 h of reperfusion, while CIN was induced in rabbits by intravenous injection of the radiocontrast medium Iopromide. Results: Treatment with parstatin 15 min before or immediately after renal ischemia attenuated the resulting renal dysfunction as demonstrated by the improved biochemical indicators (serum creatinine and fractional excretion of Na(+)) and scintigraphic analysis. The effect was dose depended and provided evidence for a more prominent protection of tubular than glomerulal function. Histopathological examination of the kidneys revealed severe renal damage, which was significantly suppressed by the parstatin. Similarly, administration of a single dose of parstatin before the induction of CIN significantly protected against the resulting renal dysfunction and histologically evidenced renal tubular injury. Conclusion: These results suggest that parstatin is able to act as nephroprotective agent and may be useful in enhancing the tolerance of the kidney against renal injury associated with clinical conditions of acute renal failure. Further investigation on the mechanism underlying the nephroprotective properties of parstatin is deemed necessary.     

Heme Oxygenase-1 Overexpression Protects Rat Livers from Ischemia/Reperfusion Injury with Extended Cold Preservation

This study analyzes the effects and mechanisms of heme oxygenase-1 (HO-1)-mediated cytoprotection in rat livers exposed to cold preservation. In the first series, rats were pretreated with cobalt protoporphyrin (CoPP) or zinc protoporphyrin (ZnPP), HO-1 inducer and antagonist, respectively. Livers were stored at 4 degrees C for 24 h, and then perfused ex vivo for 2 h. Livers pretreated with CoPP had significantly higher portal venous blood flow and increased total bile production, as compared with the ZnPP group. This correlated with histologic (Banff) criteria of hepatocyte injury/liver function. In the second series, rat livers were stored at 4 degrees C for 24 h or 40 h, and then transplanted into syngeneic recipients. After 24 h of preservation, 80% of rats bearing CoPP-pretreated liver grafts survived 21 days (vs. 50% in controls). After 40h of cold preservation, liver transplant survival at day 1, 7 and 21 for the CoPP group was: 100%, 71% and 57%, respectively (vs. 50%, 50% and 33% in controls). This correlated with improved hepatic function/histologic (Suzuki) criteria of hepatocyte injury after HO-1 overexpression (immunohistology/Western blots) by infiltrating macrophages. This study documents the potential utility of HO-1-inducing agents in preventing ischemia/reperfusion injury resulting from prolonged storage of liver transplants.     

Hematopoietic MicroRNA-126 Protects against Renal Ischemia/Reperfusion Injury by Promoting Vascular Integrity

Ischemia/reperfusion injury (IRI) is a central phenomenon in kidney transplantation and AKI. Integrity of the renal peritubular capillary network is an important limiting factor in the recovery from IRI. MicroRNA-126 (miR-126) facilitates vascular regeneration by functioning as an angiomiR and by modulating mobilization of hematopoietic stem/progenitor cells. We hypothesized that overexpression of miR-126 in the hematopoietic compartment could protect the kidney against IRI via preservation of microvascular integrity. Here, we demonstrate that hematopoietic overexpression of miR-126 increases neovascularization of subcutaneously implanted Matrigel plugs in mice. After renal IRI, mice overexpressing miR-126 displayed a marked decrease in urea levels, weight loss, fibrotic markers, and injury markers (such as kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin). This protective effect was associated with a higher density of the peritubular capillary network in the corticomedullary junction and increased numbers of bone marrow–derived endothelial cells. Hematopoietic overexpression of miR-126 increased the number of circulating Lin⁻/Sca-1⁺/cKit⁺ hematopoietic stem and progenitor cells. Additionally, miR-126 overexpression attenuated expression of the chemokine receptor CXCR4 on Lin⁻/Sca-1⁺/cKit⁺ cells in the bone marrow and increased renal expression of its ligand stromal cell-derived factor 1, thus favoring mobilization of Lin⁻/Sca-1⁺/cKit⁺ cells toward the kidney. Taken together, these results suggest overexpression of miR-126 in the hematopoietic compartment is associated with stromal cell–derived factor 1/CXCR4-dependent vasculogenic progenitor cell mobilization and promotes vascular integrity and supports recovery of the kidney after IRI.Ischemia/reperfusion injury (IRI) is a central event in such clinical conditions as AKI and in organ transplantation, and it is strongly associated with delayed graft function and long-term graft survival.^1–3 Emerging evidence indicates that the renal microvascular endothelium of the outer medullary peritubular network is the primary site of injury in the pathogenesis of ischemia-induced renal dysfunction.⁴ Following ischemia, perfusion of the peritubular capillary network is rapidly impaired as a consequence of endothelial cell (EC) swelling,⁵ impaired vasorelaxation,⁶ and increased leukocyte adhesion.⁷ In addition, microvascular destabilization initiated by the loss of EC–EC interaction⁸ and EC–pericyte interactions can lead to significant reductions in peritubular capillary density due to microvascular rarefaction.^8,9 The resulting loss in renal perfusion can further exacerbate medullary ischemia and drive the development of interstitial fibrosis by stimulation of profibrogenic factors, such as TGF-β.¹⁰ As a consequence, integrity of the peritubular capillary network is a key determinant for the preservation of renal function. Indeed, clinical biopsy studies have shown an association between loss of tubular structure and function on the one hand and capillary rarefaction on the other.^11,12Because of their limited replicative capacity, renal ECs are thought to be insufficiently capable to completely repair the injured endothelium of the peritubular capillary plexus after IRI.^13,14 Therefore, current therapeutic strategies to prevent microvascular loss have focused on the prevention of pericyte perturbation to reduce capillary rarefaction.^15–18 However, once rarefaction has occurred, these strategies may fail to induce sufficient revascularization required to reverse renal dysfunction.¹⁹ In search of ways to augment neovascularization in the injured kidney, many laboratories have investigated the biology and therapeutic use of circulating vascular progenitor cells originating from the bone marrow (BM) compartment.²⁰ These progenitor cells were shown to incorporate into the injured microvasculature in experimental models for GN,²¹ ischemic nephropathy,^22,23 and interstitial fibrosis.²⁴ This phenomenon has been particularly observed when extensive or repetitive endothelial injury occurs, for example in kidney transplantation.²⁵ Microvascular incorporation of BM-derived progenitor cells has been linked to preservation of the vasculature because they may serve as an alternative cellular source to facilitate re-endothelialization.²⁶ In addition, the CXCR4⁺ fraction of progenitor cells is mobilized to the ischemic kidney by local secretion of the chemokine stromal cell–derived factor-1 (SDF-1)^23,27 and has been shown to exert renoprotective effects in a paracrine fashion.Phosphoinositide 3-kinase (PI3K)/AKT signaling in progenitor cells plays a critical role in mobilization of progenitors from the BM via the SDF-1/CXCR4 axis²⁸ and their subsequent differentiation toward vascular cells.²⁹ MicroRNA-126 (miR-126) is a key regulator of PI3K/AKT signaling by direct targeting of the negative regulator PI3K regulatory subunit 2 (PI3KR2/p85-β) and targets genes that play key roles in angiogenesis and inflammation.^29–31 In addition, miR-126 was shown to coregulate the expansion and mobilization of hematopoietic stem cells and progenitor cells.^32,33 We hypothesized that miR-126 overexpression in the hematopoietic compartment of mice can enhance the vasoprotective potential of these progenitors and that this will translate to decreased renal injury.     

Bcl-2 Protects Tubular Epithelial Cells From Ischemia/Reperfusion Injury by Dual Mechanisms

Ischemia/reperfusion (I/R) injury, which induces extensive loss of tubular epithelial cells, is associated with delayed graft function following kidney transplantation. Recent reports have suggested that cell death by I/R injury occurs by autophagy, a cellular degradation process responsible for the turnover of unnecessary or dysfunctional organelles and cytoplasmic proteins, as well as by apoptosis. Recently, we demonstrated that overexpression of the anti-apoptotic factor, Bcl-2, inhibited tubular apoptosis and subsequent tubulointerstitial damage after I/R injury. Autophagy is also observed in cells undergoing cell death in several diseases. Therefore, we hypothesized that increased Bcl-2 protein may protect tubular epithelial cells by suppressing autophagy and inhibiting apoptosis. In the present study, a transgenic mouse model (LC3-GFP TG) in which autophagosomes are labeled with LC3-GFP and Bcl-2/LC3-GFP double transgenic mice (Bcl-2/LC3-GFP TG) were used to examine the effect of Bcl-2 on I/R-induced autophagy. I/R injury, which is associated with marked disruption of normal tubular morphology, promoted the formation of LC3-GFP dots, representing extensively induced autophagosomes. On electron microscopy, the autophagosomes contained mitochondria in I/R-injured tubular epithelial cells. In contrast, Bcl-2 augmentation suppressed the formation of autophagosomes and there was less tubular damage. In conclusion, Bcl-2 augmentation protected renal tubular epithelial cells from I/R injury by suppressing autophagosomal degradation and inhibiting tubular apoptosis.     

Fatty Acid Synthase Blockade Protects Steatotic Livers from Warm Ischemia Reperfusion Injury and Transplantation

Cerulenin has been shown to reduce body weight and hepatic steatosis in murine models of obesity by inhibiting fatty acid synthase (FAS). We have shown that attenuating intrahepatocyte lipid content diminished the sensitivity of ob/ob mice to ischemia/reperfusion injury and improved survival after liver transplantation. The mechanism of action is by inhibition of fatty acid metabolism by downregulating PPARalpha, as well as mitochondrial uncoupling protein 2 (UCP2), with a concomitant increase in ATP. A short treatment course of cerulenin prior to I/R injury is ideal for protection of steatotic livers. Cerulenin opens the potential for expanding the use of steatotic livers in transplantation.     

P-Selectin Glycoprotein Ligand-1 (rPSGL-Ig)-Mediated Blockade of CD62 Selectin Molecules Protects Rat Steatotic Liver Grafts from Ischemia/Reperfusion Injury

We examined the effects of early blockade of CD62 selectin-mediated adhesive interactions in steatotic rat liver models of ex vivo cold ischemia followed by reperfusion or transplantation by administration of P-selectin glycoprotein ligand-1 (rPSGL-Ig). In the model of cold ischemia/reperfusion, livers pretreated ex vivo with rPSGL-Ig at harvesting from obese Zucker rats showed significantly decreased portal resistance, increased bile production, and diminished hepatic endothelial neutrophil infiltration, as compared with untreated controls. Pretreatment of fatty livers with rPSGL-Ig prior to transplantation extended the survival of lean Zucker rat recipients from 40% to 90%. This effect correlated with significantly improved liver function, depressed neutrophil activity, and decreased histologic features of hepatocyte injury. Intragraft expression of CD62 P-selectin was similar in both recipient groups. rPSGL-Ig treatment decreased intragraft infiltration by CD3/CD25 cells, diminished expression of pro-inflammatory TNFalpha, IL-6, iNOS, IL-2 and IFN-gamma, without significantly affecting mRNA levels coding for anti-inflammatory IL-4. Thus, rPSGL-Ig blockade of CD62-mediated adhesive interactions protects against severe ischemia/reperfusion injury suffered otherwise by steatotic rat livers. These findings document the potential utility of rPSGL-Ig in increasing the transplant donor pool through modulation of marginal steatotic livers.     

依达拉奉对肝缺血再灌注损伤逆转作用的研究

目的研究依达拉奉影响肝脏缺血再灌注过程中TNF-α的表达情况,探讨依达拉奉对肝脏缺血再灌注损伤的逆转作用。方法将80只Wistar大鼠编号,根据计算机产生随机数字,前40为一组,后40为一组,分为实验组和对照组2组,建立常温下部分肝缺血再灌注损伤动物模型。在肝脏缺血再灌注损伤开始前1 h和开始时对实验组大鼠给予依达拉奉注射液10 ml,对照组则给予同等容量的生理盐水。分别于再灌注后0、1、2及4 h测定肝脏脂质过氧化物酶(LPO)和肝脏谷草转氨酶(AST)浓度;应用RT-PCR法检测肝组织TNF-αmRNA含量,并测定肝组织和血清中TNF-α水平;应用TUNEL染色法检测缺血肝组织的细胞凋亡情况。结果再灌注后1、2及4 h,实验组大鼠肝脏LPO及AST浓度均明显低于对照组(P<0.001);实验组再灌注后1 h时肝组织TNF-αmRNA表达量、肝组织和血清TNF-α含量均明显升高且达峰值,但均明显低于对照组(P<0.05);再灌注后各时相实验组肝细胞凋亡率明显升高,但均明显低于对照组(P<0.05)。结论依达拉奉能抑制氧化应激反应,从而降低肝缺血再灌注损伤;并显著减少炎性细胞因子TNF-α的产生,抑制炎性反应的发生,减少肝细胞的凋亡。     

Alloimmune Activation Enhances Innate Tissue Inflammation/Injury in a Mouse Model of Liver Ischemia/Reperfusion Injury

The deleterious sensitization to donor MHC Ags represents one of the most challenging problems in clinical organ transplantation. Although the role of effector/memory T cells in the rejection cascade has been extensively studied, it remains unknown whether and how these ‘Ag‐specific’ cells influence host innate immunity, such as tissue inflammation associated with ischemia and reperfusion injury (IRI). In this study, we analyzed how allogeneic skin transplant (Tx) affected the sequel of host's own liver damage induced by partial warm ischemia and reperfusion. Our data clearly showed that allo‐Tx recipients had increased inflammatory response against IR insult in their native livers, as evidenced by significantly more severe hepatocelluar damage, compared with syngeneic Tx recipient controls, and determined by serum ALT levels, liver histology (Suzuki's score) and intrahepatic proinflammatory gene inductions (TNF‐α, IL‐1β and CXCL10). The CD4 T cells, but neither CD8 nor NK cells, mediated the detrimental effect of allo‐Ag sensitization in liver IRI. Furthermore, CD154, but not IFN‐γ, was the key mechanism in allo‐Tx recipients to facilitate IR‐triggered liver damage. These results provide new evidence that alloreactive CD4 T cells are capable of enhancing innate tissue inflammation and organ injury via an Ag‐nonspecific CD154‐dependent but IFN‐γ independent mechanism.