大鼠肾脏缺血再灌注损伤中垂体中叶素及降钙素受体样受体的表达

目的 观察垂体中叶素（IMD）及其受体降钙素受体样受体（CRLR）在大鼠肾脏缺血再灌注损伤中的表达变化。 方法 将健康雄性Wistar大鼠随机分为假手术组和手术组,夹闭大鼠双侧肾动脉制作肾脏缺血再灌注损伤（IRI）模型,于缺血再灌注后0、6、12、24、48、72 h 6个时间点各取6只大鼠,留取血清及肾组织标本,对肾脏病理损伤评分并行半定量分析; Western印迹法半定量分析肾组织IMD及其受体CRLR的表达变化;放射免疫法检测血浆中IMD的表达变化。 结果 手术组大鼠发生了急性肾小管坏死（ATN）,以缺血再灌注48 h时病理损伤最重。与假手术组比较,IMD及CRLR在缺血再灌注12、24、48、72 h表达显著增高（均P < 0.01）;血浆中IMD在缺血再灌注12、48、72 h表达显著增高（均P < 0.05）。 结论 IMD及受体CRLR在大鼠肾脏缺血再灌注损伤中表达增加,血浆中IMD表达上调,提示其可能在肾脏缺血再灌注损伤病理生理过程中发挥作用。     

中性粒细胞明胶酶相关脂质运载蛋白对大鼠肾脏缺血再灌注损伤肾小管上皮细胞的保护作用

目的 探讨中性粒细胞明胶酶相关脂质运载蛋白(NGAL)对大鼠缺血再灌注损伤肾脏肾小管上皮细胞凋亡的保护作用及机制.方法 建立大鼠肾脏缺血再灌注模型,雄性SD大鼠随机分为对照组、缺血再灌注模型组、NGAL组 ;HE染色观察3组大鼠肾组织病理变化 ;TUNEL法检测肾小管上皮细胞凋亡 ;实时定量PCR、Western印迹法检测凋亡蛋白fas、bcl-2的表达变化.结果 与缺血再灌注模型组比较,NGAL组肾小管上皮细胞凋亡数量显著减少[(8.6±3.4)/HP比(20.8±3.7)/HP,P＜0.05] ;NGAL组肾组织fas mRNA(2.34±0.51比6.84±2.34,P＜0.05)、fas蛋白(0.65±0.05比0.95±0.08,P＜0.05)表达显著下调,bcl-2蛋白(0.33±0.05比0.24±0.03,P＜0.05)表达显著上调,但bcl-2 mRNA表达无明显改变.结论 NGAL对大鼠缺血再灌注损伤肾小管上皮细胞有保护作用,其作用可能与减少细胞凋亡、改变凋亡蛋白的表达有关.     

The Effects of Medical Ozone Therapy on Renal Ischemia/Reperfusion Injury

Introduction: This study was designed to investigate the possible beneficial effects of medical ozone therapy (OT), known as an immunomodulator and antioxidant, on the renal function, morphology, and biochemical parameters of oxidative stress in kidneys subjected to ischemia/reperfusion injury (IRI). Materials and methods: Thirty male Sprague–Dawley rats were classified into three groups: control, renal IRI, and renal IRI + OT. The IRI group was induced by bilateral renal ischemia for 60 min, followed by reperfusion for 6 h. After reperfusion, the kidneys and blood of rats were obtained for histopathologic and biochemical evaluation. Results: Renal IRI increased the tissue oxidative stress parameters (lipid peroxidation, protein oxidation, and nitrite plus nitrate) and decreased the antioxidant enzyme activities (superoxide dismutase and glutathione peroxidase). The serum neopterin levels showed correlation with oxidative stress parameters. All these parameters were brought to control values in the treatment group. Histopathologically, the kidney injury in the treatment group was significantly lesser than in the renal IRI group. Conclusions: Our results clearly showed that OT has beneficial effect to protect kidney against IRI. The serum neopterin levels might be used as a marker to detect the degree of renal IRI.     

钴原卟啉诱导血红素氧合酶-1高表达减轻大鼠肾脏缺血再灌注损伤

目的 探讨钴原卟啉(CoPP)诱导血红素氧合酶-1(HO-1)高表达对大鼠肾脏缺血再灌注损伤(IRI)的影响及其机理.方法 以Wistar大鼠为实验对象,CoPP组分别于左肾血流阻断前48 h和24 h腹腔注射CoPP 2.5 mg/kg,然后阻断左肾血流47 min,恢复左肾血流的同时切取大鼠右肾,采用免疫组织化学染色和免疫印迹法检测其HO-1的表达.在再灌注24 h后,处死大鼠,取其下腔静脉血和左肾,测定血肌酐(Cr)和尿素氮(BUN)浓度,观察肾组织学变化,检测肾组织中HO-1的表达.IRI组除不用CoPP处理外,其余同CoPP组.CoPP组和IRI组另有部分大鼠的血流阻断时间延长至80 min,恢复血流后不处死,观察14 d,记录其存活情况.结果 IRI组血清Cr及BUN分别为(134.37±24.26)μmol/L和(30.10±3.09)mmol/L,明显高于CoPP组的(48.92±12.92)μmol/L和(13.99±5.00)mmol/L(P＜0.05).IRI组肾小管细胞大片坏死,管型形成,与之相比,CoPP组肾小管坏死范围稍小,但肾小管病变范围仍较广泛,肾小管上皮细胞多处于水变性阶段,"缺血样"肾小球减少,管型形成较少.缺血前及再灌注24 h后,CoPP组的肾组织中HO-1均为高表达,主要位于肾间质的毛细血管处,IRI组再灌注24 h后也见肾组织中HO-1为高表达.术后14 d内,IRI组的6只大鼠中有4只死亡,而CoPP组的5只大鼠全部存活,两组大鼠存活率的差异有统计学意义(P＜0.05).结论 缺血前使用CoPP可减轻大鼠肾脏缺血再灌注损伤,该保护作用可能是通过CoPP诱导肾脏高表达HO-1来实现的.     

Class‐Specific Histone/Protein Deacetylase Inhibition Protects Against Renal Ischemia Reperfusion Injury and Fibrosis Formation

Renal ischemia‐reperfusion injury (IRI) is a common cause of renal dysfunction and renal failure. Histone/protein deacetylases (HDACs) regulate gene accessibility and higher order protein structures and may alter cellular responses to a variety of stresses. We investigated whether use of pan‐ and class‐specific HDAC inhibitors (HDACi) could improve IRI tolerance in the kidney. Using a model of unilateral renal IRI, we investigated early renal function after IRI, and calculated fibrosis after IRI using an automated scoring system. We found that pan‐HDAC inhibition using trichostatin (TSA) yielded significant renal functional benefit at 24–96 hours (p < 0.001). Treated mice developed significantly less fibrosis at 30 days (p < 0.0004). Class I HDAC inhibition with MS‐275 yielded similar effects. Protection from fibrosis formation was also noted in a cold ischemia transplant model (p < 0.008) with a trend toward improved cold ischemic survival in TSA‐treated mice. These effects were not accompanied by induction of typical ischemic tolerance pathways or by priming of heat shock protein expression. In fact, heat shock protein 70 deletion or overexpression did not alter renal ischemia tolerance. Micro‐RNA 21, known to be enhanced in vitro in renal tubular cells that survive stress, was enhanced by treatment with HDACi, pointing to possible mechanism.     

Effects of histamine and the h2 receptor antagonist ranitidine on ischemia-induced acute renal failure: involvement of IL-6 and vascular endothelial growth factor

BACKGROUND: Vascular endothelial growth factor (VEGF) exerts cytoprotective, antiapoptotic and proangiogenic effects; its synthesis is induced by hypoxia, several cytokines and histamine. The effects of histamine and the H2 receptor antagonist ranitidine on renal VEGF and IL-6 synthesis were investigated in a well-established rat model of renal ischemia/reperfusion injury. METHODS: Following 7 days of pretreatment with histamine (H group; n = 12), ranitidine (R group; n = 10) or vehicle (controls; n = 13), the left vascular pedicle was clamped for 50 min in uninephrectomized male rats and survival assessed in the different treatment groups. Additionally, renal IL-6 mRNA expression, as well as VEGF mRNA and protein abundance were measured in the three treatment groups following pretreatment only, 2 and 16 h after 50 min of renal ischemia (n = 6/group/timepoint). RESULTS: Ranitidine significantly increased, while histamine significantly decreased survival following renal ischemia. Renal IL-6 mRNA expression increased 2 h after reperfusion in all groups and decreased thereafter, with the lowest level observed in the R group. While VEGF mRNA did not change in controls, histamine increased, whereas ranitidine decreased its expression during the follow-up. Two hours after ischemia a twofold increase in renal VEGF protein abundance was observed in controls and the H group and significantly higher values were noted in the R group at this time point. A further increase in VEGF protein was only present in the H group 16 h after reperfusion. CONCLUSION: These results indicate an important role of histamine in kidney damage following renal ischemia. The beneficial effects of ranitidine were partly mediated by decreased IL-6 and VEGF mRNA expression and significant early increase in renal VEGF abundance.     

Carvedilol protects tubular epithelial cells from ischemia–reperfusion injury by inhibiting oxidative stress

Objectives: Renal ischemia–reperfusion injury (IRI), leading to acute kidney injury, is a frequent complication with renal transplantation and it is associated with graft function. Its pathogenesis involves ischemia, vascular congestion and reactive oxygen metabolites. Carvedilol is an antihypertensive drug with potent anti‐oxidant properties. In this study we investigated the protective effects of carvedilol in a rat renal IRI model. Methods: Twenty‐four rats were randomized into sham, untreated control and carvedilol (2 mg/kg 30 min before surgery and 12 hr after reperfusion) treatment groups and were subjected to 60 min of left renal ischemia followed by reperfusion at 24, 48, 96 and 168 hr. Results: Treatment with carvedilol significantly decreased plasma creatinine levels after IRI (up to 168 hr) compared to controls (P < 0.001), suggesting an improvement in renal function. Histopathological analysis revealed decreased IRI‐induced damage in kidneys from carvedilol‐treated rats. A significant increase in the expression levels of Cu/Zn superoxide dismutase and reduction of 8‐hydroxydeoxyguanosine and apoptosis levels (P < 0.005) suggested a protective effect after treatment with carvedilol. Conclusions: Our findings suggest that carvedilol ameliorates IRI resulting in improved renal function.     

Neutrophil gelatinase-associated lipocalin protects renal tubular epithelial cell in ischemic/reperfusion injury rats via apoptosis-regulating proteins

We investigated the role of neutrophil gelatinase-associated lipocalin (NGAL) on renal tubular epithelial cell in the renal ischemia/reperfusion injury (IRI) rats. Male Sprague-Dawley rats were randomly assigned to three groups. The control group (n = 5) underwent left nephrectomy. The ischemia/reperfusion (I/R) + normal saline (NS) (n = 5) and I/R + NGAL groups (n = 5) were subjected to 45 min right renal ischemia followed by 48 h of reperfusion after left nephrectomy. The pathological changes of kidney tissues were investigated using hematoxylin-eosin staining; renal tubular epithelial cell apoptosis was detected using terminal dUTP nick-labeling method; expression of apoptosis-regulating protein Fas and Bcl-2 was measured using real-time polymerase chain reaction, Western blot, and immunohistochemical staining. Compared with I/R + NS group, kidney tissues from I/R + NGAL group revealed reduced histological damage and a decreased number of renal tubular epithelial cell apoptosis (9.2 ± 2.53 nuclei or 4.0 ± 0.7 per high-power field vs. 20.3 ± 3.7 nuclei or 8.1 ± 0.3 per high-power field); rats with NGAL showed downregulated fas mRNA (2.34 ± 0.51 vs. 6.84 ± 2.34), fas protein (0.65 ± 0.05 vs. 0.95 ± 0.08), and upregulated bcl-2 protein (0.33 ± 0.05 vs. 0.24 ± 0.03). The results had statistical significance (p < 0.05). We think NGAL could protect against renal IRI and might be related to decreasing tubular epithelial cell apoptosis via adjusting the expression of apoptosis-regulating proteins.     

Beneficial Effects of N-Acetylcysteine and Ebselen on Renal Ischemia/Reperfusion Injury

Abstract

Introduction: It has been demonstrated that peroxynitrite accompanies acute renal ischemia and contributes to the pathophysiology of renal damage. Therefore, we aimed to investigate the roles of N-acetylcysteine (NAC), a well-known powerful antioxidant, and ebselen (E), a scavenger of peroxynitrite, on renal injury induced by renal ischemia/reperfusion injury (IRI) of rat kidney. Materials and methods: Forty male Sprague–Dawley rats were divided into five groups: sham, renal IRI, renal IRI+NAC, renal IRI+E, and renal IRI+NAC+E. IR injury was induced by 60 min of bilateral renal ischemia followed by 6 h of reperfusion. After reperfusion, kidneys and blood samples were obtained for histopathological and biochemical evaluations. Results: Renal IR resulted in increased malondialdehyde and nitrite/nitrate levels suggesting increased lipid peroxidation and peroxynitrite production and decreased superoxide dismutase and glutathione peroxidase activities. Both NAC and E alone significantly decreased malondialdehyde and nitrite/nitrate levels and increased superoxide dismutase and glutathione peroxidase activities. Additionally in the renal IRI+NAC+E group, all biochemical results were quite close to those of sham group. Histopathologically, the kidney injury in rats treated with combination of NAC and E was found significantly less than the other groups. Conclusions: Both NAC and E are able to ameliorate IRI of the kidney by decreasing oxidative and nitrosative stresses and increasing free radical scavenger properties. Additionally, combination of NAC and E prevents kidney damage more than when each drug is used alone, suggesting that scavenging peroxynitrite nearby antioxidant activity is important in preventing renal IRI.     

肾缺血再灌注损伤后miR-210及其靶基因的变化

目的 观察缺血再灌注损伤(IRI)对小鼠肾脏mir-210及其靶基因Ephrin-A3表达的影响.方法 将10只成年昆明雌鼠随机分为缺血再灌注组(IR)、假手术组(Sham),每组5只.IR组完全阻断双肾蒂40 min后恢复血流,Sham组暴露左右双肾40 min后关闭腹腔.每组在手术4 h后取肾组织标本,采用实时定量聚合酶链反应(PCR)检测mir-210表达水平;RT-PCR和酶标免疫组织化学染色法检测Ephrin-A3基因和蛋白的表达情况.结果 IR组miR-210表达水平明显上升(P<0.05).因miR-210对靶基因Ephrin-A3的负向调节作用,PCR结果显示IR组Ephrin-A3基因表达水平明显高于Sham组(P<0.01).Ephrin-A3蛋白主要表达在肾小管上皮细胞胞质中,Sham组Ephrin-A3蛋白表达水平较IR组明显升高(P<0.01).结论 肾缺血再灌注损伤明显影响miR-210及其靶基因Ephrin-A3的表达,miR-210表达的变化可能与肾缺血再灌注损伤的修复有关.     

intermedin对肾脏缺血再灌注损伤后血管再生相关因子表达的影响

目的 观察intermedin（IMD）对肾脏缺血再灌注损伤(IRI)后血管生长相关因子缺血诱导因子1α（HIF-1α）、血管内皮生长因子（VEGF）和血管生成素受体Tie-2表达的影响。探讨intermedin对肾脏IRI的修复作用。 方法 Wistar大鼠按随机数字表法分为4组：假手术组、IRI组、转空质粒组、转IMD质粒组。大鼠右肾切除后1周,用超声微泡造影剂介导的基因转染方法将大鼠IMD真核表达质粒转染大鼠肾脏,转染成功后夹闭左肾动脉45 min制作肾脏IRI模型。分别于再灌注1 d、2 d、3 d、4 d、7 d和14 d后留取肾组织标本,采用RT-PCR检测HIF-1α、VEGF和Tie-2的mRNA表达;Western印迹法检测肾组织VEGF的蛋白表达;ELISA检测HIF-1α和Tie-2的蛋白表达。 结果 与假手术组相比,IRI组HIF-1α mRNA和蛋白质表达于再灌注后1 d达到高峰,2 d时持续高表达,3 d时表达开始明显下降,4 d、7 d和14 d时接近于假手术组;VEGF和Tie-2表达均于再灌注后1 d开始增加,2 d达到高峰,3 d开始下降,4 d、7 d和14 d接近于假手术组;转IMD质粒组大鼠肾组织HIF-1α、VEGF和Tie-2 mRNA和蛋白质的表达于再灌注后1 d、2 d、3 d和4 d显著高于同时间点的IRI组大鼠（均P ＜ 0.05）,并且均于再灌注后1 d表达达到高峰,2～3 d持续表达,4 d开始下降,7 d和14 d的表达无明显改变。上述各指标在转空质粒和IRI组之间的表达差异无统计学意义。 结论 肾脏局部高表达的IMD不仅促进了肾脏IRI后血管再生相关因子HIF-1α、VEGF和Tie-2的表达,而且使表达高峰提前并延长了表达时间,可能参与了肾脏组织的修复和再生。     

纤维蛋白肽Bβ15～42对大鼠缺血再灌注损伤肾脏局部炎性反应的影响

目的 观察纤维蛋白肽Bβ15～42(the fibrin-derived peptide Bβ15-42,FgBβ15～42肽)对大鼠肾脏缺血再灌注损伤(IRI)后肾脏局部炎性反应的影响并探讨其机制.方法 将SD大鼠随机分成假手术组(Sham组)、IRI组、阴性治疗组和FgBβ15 ～ 42肽治疗组.Sham组:分离肾动脉后关闭腹腔;IRI组:采用双侧肾动脉夹闭的方法制作肾脏IRI模型;阴性治疗组:于肾脏再灌注后立即尾静脉注射随机肽段3.6 mg/kg; FgBβ15～42肽治疗组:于肾脏再灌注后立即尾静脉注射FgBβ15～ 42肽3.6 mg/kg.后3组按照再灌注24h、48 h分为两个亚组,Sham组与各亚组均为8只大鼠.常规生化法检测肾功能;HE、PAS染色观察肾脏组织学改变;免疫组化、实时荧光定量PCR法及Western印迹检测肾组织白细胞介素1β(IL-1β)、细胞间黏附分子1(ICAM-1)的mRNA及蛋白表达.结果 与Sham组相比,IRI组的Scr和BUN水平均显著增加(均P ＜0.05),肾小管及间质病理损伤显著,以再灌注48 h更为明显;与IRI组相比,FgBβ15～ 42肽治疗组Scr和BUN显著下降(均P＜0.05),小管间质损伤程度明显减轻(P＜0.05).与Sham组相比,IRI组IL-1β和ICA M-1的mRNA和蛋白水平于再灌注24h显著上升,48 h稍微下降,但仍维持在较高水平;FgBβ15～ 42肽治疗组大鼠肾组织IL-1β和ICAM-1的表达于再灌注24h、48 h显著低于同时间点的IRI组(均P＜0.05),但仍明显高于Sham组.上述各指标在阴性治疗组和IRI组之间的表达差异无统计学意义.结论 FgBβ15～42肽对肾脏IRI具有保护作用,其作用机制可能与其减少炎性因子IL-1β、黏附分子ICAM-1的表达有关.     

Sclerosing Encapsulating Peritonitis in an Anti-HCV-Positive Patient on Chronic Ambulatory Peritoneal Dialysis

We investigated the role of neutrophil gelatinase-associated lipocalin (NGAL) on renal tubular epithelial cell in the renal ischemia/reperfusion injury (IRI) rats. Male Sprague–Dawley rats were randomly assigned to three groups. The control group (n = 5) underwent left nephrectomy. The ischemia/reperfusion (I/R) + normal saline (NS) (n = 5) and I/R + NGAL groups (n = 5) were subjected to 45 min right renal ischemia followed by 48 h of reperfusion after left nephrectomy. The pathological changes of kidney tissues were investigated using hematoxylin–eosin staining; renal tubular epithelial cell apoptosis was detected using terminal dUTP nick-labeling method; expression of apoptosis-regulating protein Fas and Bcl-2 was measured using real-time polymerase chain reaction, Western blot, and immunohistochemical staining. Compared with I/R + NS group, kidney tissues from I/R + NGAL group revealed reduced histological damage and a decreased number of renal tubular epithelial cell apoptosis (9.2 ± 2.53 nuclei or 4.0 ± 0.7 per high-power field vs. 20.3 ± 3.7 nuclei or 8.1 ± 0.3 per high-power field); rats with NGAL showed downregulated fas mRNA (2.34 ± 0.51 vs. 6.84 ± 2.34), fas protein (0.65 ± 0.05 vs. 0.95 ± 0.08), and upregulated bcl-2 protein (0.33 ± 0.05 vs. 0.24 ± 0.03). The results had statistical significance (p < 0.05). We think NGAL could protect against renal IRI and might be related to decreasing tubular epithelial cell apoptosis via adjusting the expression of apoptosis-regulating proteins.     

基于NOD2信号通路探讨NADPH抑制剂对肾脏缺血再灌注损伤的影响

目的基于抑制核苷酸寡聚化结构域蛋白-2(nucleotide-binding oligomerization domain-2,NOD2)信号通路,探讨NADPH抑制剂对大鼠肾脏缺血再灌注损伤(ischemia/reperfusion injury,IRI)的影响及作用机制。方法将雄性Wistar大鼠切除右肾,并随机分为4组:①肾脏缺血再灌注(I/R)组:给予等量生理盐水预处理后夹闭左肾动脉制备IRI模型;②I/R组+氯化二碘联苯(diphenylene iodonium,DPI)组:给予DPI预处理后夹闭左肾动脉制备IRI模型;③I/R组+4-羟基-3甲氧基苯乙酮(4-hydroxy-3-methoxyacetophenone,Apocynin)组:给予Apocynin预处理后夹闭左肾动脉制备肾脏IRI模型;④假手术(Sham)组:给予等量生理盐水处理后不予夹闭左肾动脉。试验结束24 h后收集各组大鼠血及肾组织标本,采用Western blot法分别对NOD2、核因子κB(nuclear factor-κB,NF-κB)、半胱氨酸蛋白酶(Caspase-1)的表达进行检测;采用实时定量PCR法对NOD2 mRNA的表达进行检测;采用HE染色法观察肾脏组织学改变;采用免疫组织化学法检测肾组织炎症因子IL-1β的表达。结果与Sham组比较,I/R组的大鼠肾组织NOD2、NF-κB蛋白、Caspase-1表达显著增加(P<0.05);NOD2 mRNA表达显著增加(P<0.05);I/R组肾脏病理表现为肾小管上皮细胞水肿、坏死,脱落于管腔,肾间质炎性细胞浸润,肾小管损伤评分明显增加(P<0.05)。与I/R组相比,I/R+Apocynin组和I/R+DPI组的NOD2、NF-κB蛋白、Caspase-1表达均显著减少(P<0.05),NOD2 mRNA表达显著减少(P<0.05),肾脏病理显示急性肾小管坏死程度减轻,肾小管损伤评分显著减低(P<0.05)。结论抑制氧化应激可通过阻断NOD2受体信号通路来减轻肾脏IRI过程。     

缺血再灌注损伤后大鼠肾小管上皮细胞有机阴离子转运蛋白的表达及其功能

目的 探讨大鼠肾脏缺血再灌注损伤（IRI）后肾小管上皮细胞有机阴离子转运蛋白(OAT)1和3表达及功能变化。 方法 建立大鼠IRI模型，于术后第1、2、4、6天取血及肾组织标本。常规测定Scr浓度及观察肾脏组织形态。分别用免疫组织化学和Western印迹检测肾脏组织OAT1和3蛋白表达。毛细血管电泳仪检测肾脏对氨基马尿酸（PAH）排泄率。钼蓝分光光度法测定基侧膜钠-钾-ATP酶（Na＋-K＋-ATP酶）活性。另设假手术组作对照。 结果 模型组大鼠术后第1天肾小管上皮细胞坏死脱落，管腔内可见大量管型，刷状缘消失，基底膜裸露；Scr显著升高。与假手术组比较，模型组大鼠肾组织匀浆及肾小管上皮细胞OAT1和OAT3表达增强；PAH排泄率降低[(0.53±0.15)比(4.00 ± 0.67) ml/min, P < 0.01]；肾小管上皮细胞基侧膜Na＋-K＋-ATP酶活性降低[(9.33±1.37)比(15.07±0.15) μmol Pi&#8226;(mg蛋白)-1&#8226;h-1, P < 0.01]。损伤后第2天及第4天，损伤的肾小管逐渐修复，OAT1和3表达逐渐降低，PAH排泄率和Na＋-K＋-ATP酶活性逐渐增加；至第6天时肾小管基本恢复正常。 结论 急性肾缺血再灌注导致大鼠肾小管上皮细胞可逆性损伤，损伤初期肾小管OAT1和OAT3 表达增强，转运有机阴离子功能下降，PAH排泄减少。Na＋-K＋-ATP酶活性低下是OAT功能降低的主要机制之一。