Endogenous interleukin-6 enhances the renal injury, dysfunction, and inflammation caused by ischemia/reperfusion

Here, we investigate the effects of renal ischemia/reperfusion (I/R) on the degree of renal injury, dysfunction, and inflammation in interleukin (IL)-6 knockout (IL-6(-/-)) mice and mice administered a monoclonal antibody against IL-6. IL-6(-/-) mice were subjected to bilateral renal artery occlusion (30 min) and reperfusion (24 h). At the end of experiments, indicators and markers of renal dysfunction, injury, and inflammation were measured. Kidneys were used for histological evaluation of renal injury. Renal expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and P-selectin, as well as nitration of proteins in the kidney, were determined using immunohistochemistry. In addition, wild-type mice were pretreated (24 and 1 h before ischemia) with an IL-6 antibody to mimic the effects that would be seen in IL-6(-/-) mice. IL-6(-/-) mice and wild-type mice administered the IL-6 antibody demonstrated significantly reduced plasma urea and creatinine levels, indicating reduction of renal dysfunction caused by I/R. Neutrophil infiltration was also significantly reduced in IL-6(-/-) mice and wild-type mice administered the IL-6 antibody subjected to renal I/R. Proinflammatory cytokines (tumor necrosis factor-alpha and IL-1beta) in renal tissues were significantly attenuated in IL-6(-/-) mice to levels seen in wild-type mice. IL-6(-/-) mice demonstrated reduced histological evidence of tubular injury and markedly reduced immunohistochemical evidence of ICAM-1, P-selectin, and nitrotyrosine when subjected to renal I/R. We propose that endogenous IL-6 enhances the degree of renal injury, dysfunction, and inflammation caused by I/R of the kidney by promoting the expression of adhesion molecules and subsequent oxidative and nitrosative stress.     

Intercellular adhesion molecule-1-deficient mice are protected against ischemic renal injury.

Studies in the rat have pointed to a role for intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of acute tubular necrosis. These studies used antibodies, which may have nonspecific effects. We report that renal ICAM-1 mRNA levels and systemic levels of the cytokines IL-1 and TNF-alpha increase 1 h after ischemia/ reperfusion in the mouse. We sought direct proof for a critical role for ICAM-1 in the pathophysiology of ischemic renal failure using mutant mice genetically deficient in ICAM-1. ICAM-1 is undetectable in mutant mice in contrast with normal mice, in which ICAM-1 is prominent in the endothelium of the vasa recta. Mutant mice are protected from acute renal ischemic injury as judged by serum creatinine, renal histology, and animal survival . Renal leukocyte infiltration, quantitated morphologically and by measuring tissue myeloperoxidase, was markedly less in ICAM-1-deficient than control mice. To evaluate whether prevention of neutrophil infiltration could be responsible for the protection observed in the mutant mice, we treated normal mice with antineutrophil serum to reduce absolute neutrophil counts to < 100 cells/mm3. These neutrophil-depleted animals were protected against ischemic renal failure. Anti-1CAm-1 antibody protected normal mice against renal ischemic injury but did not provide additional protection to neutrophil-depleted animals. Thus, ICAM-1 is a key mediator of ischemic acute renal failure likely acting via potentiation of neutrophilendothelial interactions.     

Endothelial nitric oxide synthase protects the post-ischemic liver: potential interactions with superoxide.

Hepatic ischemia and reperfusion (I/R) continues to represent a significant cause of post-transplant liver failure. The roles that certain free radicals including nitric oxide (NO) and superoxide (O(2)(-)) play in this process are not well understood. The present study was designed to assess the role of endothelial cell nitric oxide synthase (eNOS) in I/R-induced liver injury in a murine model of hepatic I/R. Forty five minutes of partial (70%) hepatic ischemia followed by 3 and 6 h of reperfusion resulted in a significant increase in liver injury which occurred in the absence of neutrophil infiltration. eNOS-deficient mice displayed enhanced liver injury when compared to their wild type controls again in the absence of neutrophil infiltration. Interestingly, basal liver blood flow was significantly decreased in these mice when compared to controls though their blood flow during reperfusion was not significantly reduced from their wild type controls. Treatment of eNOS(-/-) mice with gadolinium chloride, a potent inhibitor of Kupffer cell function, but not superoxide dismutase, significantly reduced post-ischemic hepatocellular injury while either treatment protected the wild type mouse livers. Taken together, these data suggest that NO derived from eNOS may act to protect the post-ischemic liver possibly by suppression of Kupffer cell function and not by modulation of tissue perfusion. Further the data presented here would indicate that the protective effects conferred by SOD are related to its ability to increase the bioavailability of NO rather than by attenuating superoxide-dependent reactions. Data generated from these studies may prove useful in developing new drug therapies to treat the post-ischemic liver.     

Transgenic expression of human complement regulators reduces skeletal muscle ischaemia/reperfusion injury in mice

This study aimed to explore the hypothesis that activated complement components contribute significantly to I/R (ischaemia/reperfusion) injury in skeletal muscle. After 50, 70 and 90 min of tourniquet ischaemia and 24 h of reperfusion, viability of the medial gastrocnemius muscle in CBA-C57BL/6 wild-type mice, assessed histochemically by reduction of NBT (Nitro Blue Tetrazolium) dye, was 60, 21 and 8% respectively. Skeletal muscle viability after 70 min of ischaemia and 24 h of reperfusion in transgenic mice expressing a combination of human CD46, CD55 and CD59, all inhibitors of complement activation, was 45% compared with 24% in ischaemic reperfused wild-type mice (P=0.008; n=6 per group). Muscle from sham-treated transgenic mice and wild-type littermates had no significant loss of viability relative to normal contralateral gastrocnemius muscle. A significant reduction in myeloperoxidase activity (a measure of neutrophil infiltration), xanthine oxidase activity (a source of free radicals) and water content (a measure of oedema) was observed in ischaemic reperfused muscle from transgenic mice compared with ischaemic reperfused wild-type muscle (P<0.05). Haematoxylin and eosin-stained histological sections also showed less damage and less apparent leucocyte infiltration in muscles from ischaemic reperfused transgenic mice than those from wild-type animals given the same degree of injury. Muscles from sham-treated transgenic and wild-type controls were almost identical with normal muscle. It is concluded that complement activation contributes to the pathogenesis of I/R injury in murine skeletal muscle, resulting in increased neutrophil infiltration into the injured muscle, increased free radical production and vascular permeability during reperfusion, and a net detrimental effect on muscle viability.     

Elimination of C5aR prevents intestinal mucosal damage and attenuates neutrophil infiltration in local and remote organs

The complement C5a pathway has been shown to be an important mediator of inflammation and tissue injury. To further understand the role of C5a receptor (C5aR) pathway in ischemia/reperfusion (I/R) injury, and to evaluate the potential of antagonizing C5aR to protect from I/R injury, we tested the effect of eliminating C5aR using C5aR knockout (KO) mice and their wild-type (WT) littermates in a superior mesenteric artery occlusion (SMAO) intestinal I/R injury model. C5aR KO and WT mice were subjected to SMAO or sham for 45 min. After 3 h of reperfusion, the percentage of injured ileal villi was twice as high in WT mice subjected to SMAO as compared with the C5aR KO mice. In addition, the number of neutrophils was 34% higher in WT mice subjected to SMAO as compared with the C5aR KO mice. Moreover, ileum and lung myeloperoxidase activities after SMAO were significantly higher in WT than C5aR KO mice. Apoptotic cell death was induced after reperfusion in WT-SMAO and was reduced by more than 50% in C5aR KO mice. The plasma level of TNF-alpha was increased approximately 3.74-fold in WT subjected to SMAO compared with sham. In contrast, the level was increased only approximately 1.18-fold in the C5aR KO mice subjected to SMAO. In conclusion, this study demonstrates that elimination of the C5aR pathway protects the intestine from I/R injury and diminishes intestine-derived pulmonary neutrophil sequestration. Blocking C5aR may be considered as a potential therapeutic intervention for I/R injury.     

Renal-associated TLR2 mediates ischemia/reperfusion injury in the kidney

TLRs are conserved pattern recognition receptors that detect motifs of pathogens and host material released during injury. For unknown reasons, renal TLR2 mRNA is mainly expressed by tubular cells and is enhanced upon renal ischemia/reperfusion (I/R) injury. We evaluated the role of TLR2 in I/R injury using TLR2-/- and TLR2+/+ mice, TLR2 antisense oligonucleotides, and chimeric mice deficient in leukocyte or renal TLR2. Tubular cells needed TLR2 to produce significant cytokine and chemokine amounts upon ischemia in vitro. TLR2 played a proinflammatory and detrimental role in vivo after I/R injury, as reflected by a reduction in the amount of local cytokines and chemokines, leukocytes, and the level of renal injury and dysfunction in TLR2-/- mice compared with controls. Analysis of chimeric mice suggested that TLR2 expressed on renal parenchyma plays a crucial role in the induction of inflammation and injury. TLR2-antisense treatment protected mice from renal dysfunction, neutrophil influx, and tubular apoptosis after I/R injury compared with nonsense treatment. In summary, we identified renal-associated TLR2 as an important initiator of inflammatory responses leading to renal injury and dysfunction in I/R injury. These data imply that TLR2 blockade could provide a basis for therapeutic strategies to treat or prevent renal ischemic injury.     

Altered renal tubular expression of the complement inhibitor Crry permits complement activation after ischemia/reperfusion

Ischemia/reperfusion (I/R) of several organs results in complement activation, but the kidney is unique in that activation after I/R occurs only via the alternative pathway. We hypothesized that selective activation of this pathway after renal I/R could occur either because of a loss of complement inhibition or from increased local synthesis of complement factors. We examined the relationship between renal complement activation after I/R and the levels and localization of intrinsic membrane complement inhibitors. We found that loss of polarity of complement receptor 1-related protein y (Crry) in the tubular epithelium preceded activation of the alternative pathway along the basolateral aspect of the tubular cells. Heterozygous gene-targeted mice that expressed lower amounts of Crry were more sensitive to ischemic injury. Furthermore, inhibition of Crry expressed by proximal tubular epithelial cells in vitro resulted in alternative pathway-mediated injury to the cells. Thus, altered expression of a complement inhibitor within the tubular epithelium appears to be a critical factor permitting activation of the alternative pathway of complement after I/R. Increased C3 mRNA and decreased factor H mRNA were also detected in the outer medulla after I/R, suggesting that altered synthesis of these factors might further contribute to complement activation in this location.     

CD4(+) T-lymphocytes mediate ischemia/reperfusion-induced inflammatory responses in mouse liver.

The success of orthotopic liver transplantation is dependent on multiple factors including MHC tissue compatibility and ischemic/reperfusion injury. Ischemic/reperfusion (I/R) injury in the liver occurs in a biphasic pattern consisting of both acute phase (oxygen free radical mediated) and subacute phase (neutrophil-mediated) damage. Although numerous studies have given insights into the process of neutrophil recruitment after I/R injury to the liver, the exact mechanism that initiates this subacute response remains undefined. Using a T cell-deficient mouse model, we present data that suggests that T-lymphocytes are key mediators of subacute neutrophil inflammatory responses in the liver after ischemia and reperfusion. To this end, using a partial lobar liver ischemia model, we compared the extent of reperfusion injury between immune competent BALB/c and athymic nu/nu mice. Studies evaluating the extent of liver damage as measured by serum transaminases (GPT) demonstrate similar acute (3-6 h) post-I/R responses in these two mouse models. In contrast, the subacute phase (16-20 h) of liver injury, as measured by both serum GPT levels and percent hepatocellular necrosis, was dramatically reduced in T cell-deficient mice as compared with those with an intact immune system. This reduction in liver injury seen in nu/nu mice was associated with a 10-fold reduction in hepatic neutrophil infiltration. Adoptive transfer of T cell-enriched splenocytes from immune competent mice was capable of reconstituting the neutrophil-mediated subacute inflammatory response within T cell-deficient nu/nu mice. Furthermore, in vivo antibody depletion of CD4(+) T-lymphocytes in immune competent mice resulted in a reduction of subacute phase injury and inflammation as measured by serum GPT levels and neutrophil infiltration. In contrast, depletion of CD8(+) T-lymphocytes had no effect on these indexes of subacute inflammation. Kinetic analysis of T cell infiltration in the livers of BALB/c mice demonstrated a fivefold increase in the number of hepatic CD4(+) T-lymphocytes within the first hour of reperfusion with no significant change in the number of CD8(+) T-lymphocytes. In summary, these results implicate CD4(+) T-lymphocytes as key regulators in initiating I/R-induced inflammatory responses in the liver. Such findings have implications for therapy directed at the early events in this inflammatory cascade that may prove useful in liver transplantation.     

膜攻击复合物C5b-9在大鼠肝脏缺血再灌注损伤中的表达

目的探讨膜攻击复合物C5b-9在大鼠肝脏缺血再灌注损伤中的表达。方法健康雄性SD大鼠60只,180²00 g,随机分为5组(n=12):假手术组(S组)、全肝再灌组1 h组(I/R1 h组)、3 h组(I/R 3 h组)、6 h组(I/R 6 h组)和24 h组(I/R 24 h组)。制备大鼠全肝缺血再灌注模型,再灌注1、3、6、24 h末,取血测定谷丙转氨酶(ALT)和总胆红素(TBIL)浓度。取肝脏组织,检测C5b-9 mRNA和C5b-9蛋白的表达。结果与对照组比较,I/R各组大鼠ALT和TBIL水平均明显升高,I/R 3 h组达到高峰(P<0.05);I/R各组C5b-9的mRNA表达和C5b-9蛋白含量均有显著升高,且随着缺血再灌注时间的延长而逐渐升高,其中I/R 24 h组最高(P<0.05)。结论补体C5b-9参与肝缺血再灌注的损伤,尤其是在再灌注24 h时达到高峰。     

改构型和野生型aFGF对肠缺血-再灌注损伤后肝肾功能的影响

目的　观察肠缺血再灌注损伤后的肝、肾功能的变化 ,并探讨不同类型酸性成纤维细胞生长因子 (a FGF)对肠缺血再灌注损伤后的肝、肾功能的保护作用。方法　将 78只 Wistar大鼠分成假手术组 (C)、生理盐水治疗组 (R)、改构型 a FGF治疗组 (rh F)和野生型 a FGF治疗组 (wt F) ,后 3组根据再灌注时间又分为 2、6、12和 2 4 h4个时间点。阻断肠系膜上动脉 4 5 m in后松夹制备肠缺血再灌注模型。于不同时间点腹主动脉抽血 5 ml,检测肝、肾功能的变化。结果　与 C组比较 ,R组、rh F组和 wt F组肝、肾功能都有明显下降 ,以 R组下降更明显 ,rh F组次之 ,wt F组肝、肾功能恢复得最好。组织病理学检查发现 ,3组动物均有小肠绒毛脱落、黏膜下层炎细胞浸润等改变 ,其严重程度与肝、肾功能指标水平一致。结论　肠缺血再灌注损伤导致多脏器功能损伤 ,野生型、改构型 a FGF对脏器功能有明显的保护作用。     

Tubulointerstitial injury induced in rats by a monoclonal antibody that inhibits function of a membrane inhibitor of complement.

The kidney widely expresses membrane-associated complement regulatory proteins (membrane inhibitors of complement). The aim of this work was to evaluate the roles of these molecules in rat kidneys in vivo. To suppress functions of rat membrane inhibitors of complement, two mAbs, 512 and 6D1, were used. 5I2 and 6D1 inhibit functions of membrane inhibitors of complement at C3 level (rat Crry/p65) and C8/9 level (rat CD59), respectively. F(ab')2 fragment of 5I2 or 6D1 was perfused in the left kidneys, and perfusate was discarded from the renal vein. After perfusion, the left kidneys were connected to systemic circulation. In rats perfused with 5I2, mouse IgG was found in glomeruli, peritubular capillaries, vascular bundles, and tubules 15 min after recirculation. Binding of C3 and C5b-9 was evident in these areas. 1 d after perfusion with 5I2, cast formation, dilatation of tubular lumen, and tubular cell degeneration were observed. At day 4 through day 7, significant mononuclear cell infiltration and proximal tubule damage were observed. These changes were completely prevented by complement depletion. Rats perfused with 6D1 showed the binding of mouse IgG in the similar areas as 5I2, but C3 or C5b-9 deposition was not observed. Rats perfused with 6D1 or vehicle only did not show any pathology in the left kidneys. These results suggest that rat Crry/p65 plays protective roles against spontaneously occurring indiscriminate attack to tubulointerstitial tissues by autologous complement and that rat Crry/p65 is one of the important factors to maintain normal integrity of the kidney in rats.     

Cyclic arginine-glycine-aspartate attenuates acute lung injury in mice after intestinal ischemia/reperfusion

Introduction

Intestinal ischemia is a critical problem resulting in multiple organ failure and high mortality of 60 to 80%. Acute lung injury (ALI) is a common complication after intestinal ischemia/reperfusion (I/R) injuries and contributes to the high mortality rate. Moreover, activated neutrophil infiltration into the lungs is known to play a significant role in the progression of ALI. Integrin-mediated interaction is involved in neutrophil transmigration. Synthetic peptides containing an arginine-glycine-aspartate sequence compete with adhesive proteins and inhibit integrin-mediated interaction and signaling. Thus, we hypothesized that the administration of a cyclic arginine-glycine-aspartate peptide (cRGD) inhibited neutrophil infiltration and provided protection against ALI induced by intestinal I/R.

Methods

Ischemia in adult male C57BL/6 mice was induced by fastening the superior mesenteric artery with 4-0 suture. Forty-five minutes later, the vascular suture was released to allow reperfusion. cRGD (5 mg/kg body weight) or normal saline (vehicle) was administered by intraperitoneal injection 1 hour prior to ischemia. Blood, gut, and lung tissues were collected 4 hours after reperfusion for various measurements.

Results

Intestinal I/R caused severe widespread injury to the gut and lungs. Treatment with cRGD improved the integrity of microscopic structures in the gut and lungs, as judged by histological examination. Intestinal I/R induced the expression of β₁, β₂ and β₃ integrins, intercellular adhesion molecule-1, and fibronectin. cRGD significantly inhibited myeloperoxidase activity in the gut and lungs, as well as neutrophils and macrophages infiltrating the lungs. cRGD reduced the levels of TNF-α and IL-6 in serum, in addition to IL-6 and macrophage inflammatory protein-2 in the gut and lungs. Furthermore, the number of TUNEL-staining cells and levels of cleaved caspase-3 in the lungs were significantly lowered in the cRGD-treated mice in comparison with the vehicle mice.

Conclusions

Treatment with cRGD effectively protected ALI and gut injury, lowered neutrophil infiltration, suppressed inflammation, and inhibited lung apoptosis after intestinal I/R. Thus, there is potential for developing cRGD as a treatment for patients suffering from ALI caused by intestinal I/R.     

Cisplatin-induced acute renal failure is associated with an increase in the cytokines interleukin (IL)-1beta, IL-18, IL-6, and neutrophil infiltration in the kidney

We have demonstrated that caspase-1-deficient (caspase-1(-/-)) mice are functionally and histologically protected against cisplatin-induced acute renal failure (ARF). Caspase-1 exerts proinflammatory effects via the cytokines interleukin (IL)-1beta, IL-18, IL-6, and neutrophil recruitment. We sought to determine the role of the cytokines IL-1beta, IL-18, and IL-6 and neutrophil recruitment in cisplatin-induced ARF. We first examined IL-1beta; renal IL-1beta increased nearly 2-fold in cisplatin-induced ARF and was reduced in the caspase-1(-/-) mice. However, inhibition with IL-1 receptor antagonist (IL-1Ra) did not attenuate cisplatin-induced ARF. Renal IL-18 increased 2.5-fold; however, methods to inhibit IL-18 using IL-18 antiserum and transgenic mice that overproduce IL-18-binding protein (a natural inhibitor of IL-18) did not protect. Renal IL-6 increased 3-fold; however, IL-6-deficient (IL-6(-/-)) mice still developed cisplatin-induced ARF. We next examined neutrophils; blood neutrophils increased dramatically after cisplatin injection; however, prevention of peripheral neutrophilia and renal neutrophil infiltration with the neutrophil-depleting antibody RB6-8C5 did not protect against cisplatin-induced ARF. In summary, our data demonstrated that cisplatin-induced ARF is associated with increases in the cytokines IL-1beta, IL-18, and IL-6 and neutrophil infiltration in the kidney. However, inhibition of IL-1beta, IL-18, and IL-6 or neutrophil infiltration in the kidney is not sufficient to prevent cisplatin-induced ARF.     

Role of the terminal complement pathway in experimental membranous nephropathy in the rabbit.

Our recent observations of a complement-mediated, cell-independent mechanism of altered glomerular permeability in rat membranous nephropathy suggested a possible role for the terminal complement pathway in the mediation of proteinuria in certain forms of glomerular disease. To directly determine whether the membranolytic terminal complement components (C5b-C9) are involved in glomerular injury, we studied the development of proteinuria in normal and C6-deficient (C6D) rabbits, in both of which a membranous nephropathy-like lesion develops early in the course of immunization with cationized bovine serum albumin (cBSA) (pI 8.9-9.2). C6 hemolytic activity of C6D was 0.01% that of control rabbits. After 1 wk of daily intravenous injections of cBSA, proteinuria developed in 71% of controls (median 154, range 1-3,010 mg/24 h, n = 24), whereas none of C6D were proteinuric (median 6, range 2-12 mg/24 h, n = 12, P less than 0.01). After 1 wk of cBSA, both groups had qualitatively identical glomerular deposits of BSA, rabbit IgG, and C3 on immunofluorescence microscopy, predominantly subepithelial electron-dense deposits on electron microscopy, and minimal glomerular inflammatory cell infiltration of glomeruli. Glomeruli were isolated from individual animals after 1 wk of cBSA and deposits of rabbit IgG antibody were quantitated by a standardized in vitro assay using anti-rabbit IgG-125I. Rabbit IgG deposits were found to be similar in control (29.8 +/- 13.2, range 12.7-48.6 micrograms anti-IgG/2,000 glomeruli, n = 6) and C6D rabbits (32.6 +/- 13.8, range 16.8-48.8 micrograms anti-IgG/2,000 glomeruli, n = 5, P greater than 0.05). After 2 wk, coincident with a prominent influx of mononuclear cells and neutrophils, proteinuria developed in C6D rabbits. These results document, for the first time, a requirement for a terminal complement component in the development of immunologic glomerular injury. Since the only known action of C6 is in the assembly of the membrane attack complex, these observations suggest that the membranolytic properties of complement may contribute to glomerular damage.     

Protective effects of exogenous interleukin 18-binding protein in a rat model of acute renal ischemia-reperfusion injury

Ischemia-reperfusion (I/R) renal injury is considered the most common cause of acute kidney injury (AKI). The pathophysiology of I/R AKI involves a complex interplay among tubular epithelial cell injury, microcirculation dysfunction, and inflammation. Interleukin 18-binding protein (IL-18BP) is a natural inhibitor of IL-18 a cytokine that plays an important role in the pathogenesis of AKI. Therefore, we hypothesized that exogenous IL-18BP could protect against renal injuries after kidney I/R. Male Sprague-Dawley rats were divided into three groups: a sham operation group, I/R with vehicle injection, and I/R with IL-18BP injection. Rats underwent bilateral renal pedicle clamping, and IL-18BP or vehicle was administered just before reperfusion. Rats were killed 6, 24, and 72 h after reperfusion. After IL-18BP treatment, renal tubule epithelium showed reduced apoptosis and enhanced proliferation. For peritubular capillary (PTC) endothelium, apoptosis was inhibited, and there was an increase in PTC endothelium density. Macrophage infiltration was inhibited, and inflammatory cytokines were downregulated. Increased expression of vascular endothelial growth factor and decreased expression of thrombospondin 1 were also observed. Exogenous IL-18BP attenuated renal injury caused by I/R via inhibiting inflammation in the renal tissue and protecting tubular epithelium and PTC endothelium.     

肾缺血对缺氧诱导microRNAs及VEGF-NOTCH信号分子的影响

目的 观察肾缺血性损伤后,缺氧诱导microRNAs及VEGF-NOTCH信号分子的表达变化,探讨肾缺血损伤后血管新生的可能调控途径.方法 选择雄性Balb/c小鼠20只,采用夹闭双侧肾蒂方法制备急性缺血肾损伤模型,假手术组设为对照组.通过观察缺血恢复后24 h小鼠肾功能及肾组织病理学改变确定模型成功.实时定量RT-PCR检测缺血恢复后4 h,24 h时缺氧诱导miRNA-210,miRNA-92a,VEGF,Flk-1及Notch1 mRNA表达水平;Western-blot检测缺血恢复后24 h,72 h时Flk-1蛋白的变化;免疫组织化学染色检测CD31表达,判断缺血组织微血管内皮细胞增生状况.结果 肾缺血恢复24 h小鼠肌酐、尿素氮明显升高(P＜0.05),光镜下观察大量小管上皮细胞肿胀、空泡变性坏死,肾小管管腔扩张,见上皮细胞碎片和管型,表明成功建立肾缺血损伤模型.肾缺血恢复4 h,24 h后,与正常组比较,肾组织miRNA-210上调倍数分别为(2.02±0.29),(5.58±0.16);miRNA-92a的表达上调倍数分别为(3.23±0.74),(1.53±0.33),P＜0.05;VEGF,Flk-1及Notch1 mRNA表达水平均升高(P＜0.05).缺血恢复后24 h,72 h时,Flk-1蛋白水平显著升高(P＜0.05),肾组织微血管密度明显增加.结论 肾缺血性损伤后可出现代偿性血管新生,且缺氧诱导miRNA如miRNA-92a,miRNA-210表达上调,与血管新生相关的信号分子VEGF及Notch1表达均升高,提示miRNA/VEGF-Notch1可能是肾缺血性损伤后血管新生的主要调控通路,从而为探讨缺血性损伤后血管新生的机制提供了依据.

Abstract：

Objective To investigate the expression changes of microRNAs and VEGF-NOTCH in renal ischemic injury in mice, and to explore the potential mechanism associated with renal angiogenesis.Method Male Balb/c mice were subjected to a standard renal ischemia to induce acute kidney injury (AKI) after 45 min of bilateral renal artery clamping. Following 4 h, 24 h of reperfusion or sham operation, kindey tissues were collected and subjected to detect the expression changes of microRNAs which relatived with angiogenesis and VEGF, Flk-1, Notch1 mRNA by Quantitative Real-time RT-PCR. Flk-1 protein was detected by Western blotting analysis at 24 h and 72 h following Ischemia/Reperfusion(I/R) injury. The expression of CD31 was examined in tissue sections by immunohistochemistry staining, and the microvessels in ischemic region of each group were counted. Results miRNA-210 and miRNA-92a expression increased significantly, with prominent changes at 4 h and 24 h after reperfusion( P ＜ 0.05 ). VEGF and Flk-1 mRNA expression and Flk-1 protein were increased in renal I/R compared with control group respectively (P＜0.05 ).Immunohistochemistry staining results of CD31 showed a significant increase of microvessels in renal ischemic region. Conclusion This study first reported the changes in miRNAs expression in response to kidney I/R in mouse. our results implied that miRNAs may be involved in targeting VEGF-Notch pathway signaling to regulate angiogenesis after renal I/R injury. It provided novel insights into the angiogenesis mechanism of renal ischemic injury.     

Bone marrow-derived cells mobilized by granulocyte-colony stimulating factor facilitate vascular regeneration in mouse kidney after ischemia/reperfusion injury

Bone marrow-derived cells (BMDC) play crucial roles in tissue regeneration. Granulocyte-colony stimulating factor (G-CSF) mobilizes BMDC and may facilitate the repair of kidney tissues after ischemia/reperfusion (I/R) injury. The tissue protective action of resveratrol, an antioxidant, might modify the regenerating potential of BMDC in I/R renal injury. This study examined whether G-CSF and/or resveratrol affect the recruitment of BMDC into vascular endothelial cells and renal tubular cells and the kidney function after I/R injury. I/R renal injury was induced in female mice that had been lethally irradiated and transplanted with male bone marrow cells. The mice were given saline, resveratrol or G-CSF, daily for 7 days. Non-irradiated and non-bone-marrow-transplanted female mice, which underwent the same kidney injury, were included as control. White blood cell (WBC) count and serum creatinine were monitored. Immunohistologic evaluation for renal tubular cells (cytokeratin) and endothelial cells (factor VIII-related antigen), and fluorescence in situ hybridization for mouse Y chromosome were performed. Although WBC was significantly higher in the G-CSF group, there was no significant difference in creatinine levels among all groups. Factor VIII-related antigen-positive cells with a Y-chromosome signal were identified in the capillary wall between renal tubuli and most frequently seen in the G-CSF group (p < 0.0001). Resveratrol did not affect kidney recovery in this model. No cytokeratin-positive renal tubular cells having a Y-chromosome signal were identified. In conclusion, BMDC are recruited into endothelial cell in I/R renal injury without apparent renal tubular cell regeneration, and G-CSF facilitates the endothelial cell regeneration.     

Cardiotrophin-1 defends the liver against ischemia-reperfusion injury and mediates the protective effect of ischemic preconditioning

Ischemia-reperfusion (I/R) liver injury occurs when blood flow is restored after prolonged ischemia. A short interruption of blood flow (ischemic preconditioning [IP]) induces tolerance to subsequent prolonged ischemia through ill-defined mechanisms. Cardiotrophin (CT)-1, a cytokine of the interleukin-6 family, exerts hepatoprotective effects and activates key survival pathways like JAK/STAT3. Here we show that administration of CT-1 to rats or mice protects against I/R liver injury and that CT-1-deficient mice are exceedingly sensitive to this type of damage. IP markedly reduced transaminase levels and abrogated caspase-3 and c-Jun-NH2-terminal kinase activation after I/R in normal mice but not in CT-1-null mice. Moreover, the protective effect afforded by IP was reduced by previous administration of neutralizing anti-CT-1 antibody. Prominent STAT3 phosphorylation in liver tissue was observed after IP plus I/R in normal mice but not in CT-1-null mice. Oxidative stress, a process involved in IP-induced hepatoprotection, was found to stimulate CT-1 release from isolated hepatocytes. Interestingly, brief ischemia followed by short reperfusion caused mild serum transaminase elevation and strong STAT3 activation in normal and IL-6-deficient mice, but failed to activate STAT3 and provoked marked hypertransaminasemia in CT-1-null animals. In conclusion, CT-1 is an essential endogenous defense of the liver against I/R and is a key mediator of the protective effect induced by IP.     

Active site inhibited factor VIIa attenuates myocardial ischemia/reperfusion injury in mice

Summary.  Background:  Inhibition of specific coagulation pathways such as the factor VIIa-tissue factor complex has been shown to attenuate ischemia/reperfusion (I/R) injury, but the cellular mechanisms have not been explored. Objectives:  To determine the cellular mechanisms involved in the working mechanism of active site inhibited factor VIIa (ASIS) in the protection against myocardial I/R injury. Methods:  We investigated the effects of a specific mouse recombinant in a mouse model of myocardial I/R injury. One hour of ischemia was followed by 2, 6 or 24 h of reperfusion. Mouse ASIS or placebo was administered before and after induction of reperfusion. Results:  ASIS administration reduced myocardial I/R injury by more than 40% at three reperfusion times. Multiplex ligation dependent probe amplification (MLPA) analysis showed reduced mRNA expression in the ischemic myocardium of CD14, TLR-4, interleukin-1 (IL-1) receptor-associated kinase (IRAK) and IκBα upon ASIS administration, indicative of inhibition of toll-like receptor-4 (TLR-4) and subsequent nuclear factor-κB (NF-κB) mediated cell signaling. Levels of nuclear activated NF-κB and proteins influenced by the NF-κB pathway including tissue factor (TF) and IL-6 that were increased after I/R, were attenuated upon ASIS administration. After 6 and 24 h of reperfusion, neutrophil infiltration into the area of infarction was decreased upon ASIS administration. There was, however, no evidence of an effect of ASIS on apoptosis (Tunel staining and MLPA analysis). Conclusions:  We conclude that the diminished amount of myocardial I/R injury after ASIS administration is primarily due to attenuated inflammation-related lethal I/R injury, probably mediated through the NF-κB mechanism.