Mitochondrial dysfunction in the process of cisplatin-induced acute kidney injury in mice

Objective To assess the characteristics of different doses of cisplatin-induced acute kidney injury, further to understand mitochondrial dysfunction and its role in acute kidney injury (AKI). Methods Male C57BL/6J mice were first randomly divided into two groups: control group (n＝6) and AKI group (n＝12). Then, AKI group was subsequently divided into other two groups according to different dose of cisplatin (10 mg/kg or 20 mg/kg). AKI group received intraperitoneal injection of cisplatin. All mice were sacrificed after 72 h of injection. Renal biochemical function, renal pathological changes, renal injury markers, kidney mitochondrial function and structural changes were observed. Results (1) After 72 hours of injection, the AKI group performed significant kidney injury changes compared to control group, thereinto 20 mg/kg group was more serious than 10 mg/kg group. With the cisplatin dose increasing, renal function markers such as serum creatinine, urine protein gradually increased. (2)Kidney biopsy showed tubular structural damage, the formation of protein casts, kidney injury molecule-1 (KIM-1) gradually increased(P＜0.05). (3)Electron microscopy found tubular mitochondrial structural damage, mtDNA copy number decreased, the level of peroxisome proliferator-activated receptor -gamma coactivator-1alpha (PGC-1α), ATP synthase β decreased(P＜0.05), and Western blotting manifested cytochrome C was released from mitochondria to the cytoplasm. These data all exhibited significant difference between different groups(P＜0.05). Conclusions Cisplatininduces acute kidney injury in dose-dependent manner. Mitochondrial dysfunction participates in kidney injury, and is also related to the kidney pathological damage.     

Expression of apoptosis stimulating protein two of p53 in acute kidney injury induced by carbon tetrachloride in mice

Objective To investigate the expression of apoptosis stimulating protein two of p53 (ASPP2) in acute kidney injury (AKI) induced by carbon tetrachloride (CCl4) in mice. Methods Thirty-two male Balb/c mice were randomly divided into olive oil control group (control group, 8 mice) and CCl4 experimental group (experimental group, 24 mice). A mouse model of AKI was induced by a single high-dose abdominal injection of CCl4. The mice in the experimental group were sacrificed at 12 h, 24 h and 48 h after CCl4 injection. The mice in the control group were sacrificed 24 h after treatment. The serum and kidney tissue samples were collected. Serum biochemical indicators [serum creatinine (Scr) and urea nitrogen (BUN)] were measured by automatic biochemical analyzer. The pathological damage of kidney tissue was observed by hematoxylin-eosin (HE) staining and Periodate-Schiff (PAS) staining. ASPP2 positioning and expression level were observed by immunohistochemistry. The apoptosis of mouse kidney tissue was detected by in situ apoptosis. The expression of ASPP2 protein and ASPP2 mRNA in renal tissue were detected by Western blotting and quantitative real-time PCR. Results Compared with the control group, the levels of Scr and BUN were significantly increased in the experimental group (P＜0.01). Histopathology showed partial renal tubular brush margin detachment, renal tubular epithelial cell necrosis and nuclear disintegration in the experimental group. TUNEL staining showed that the apoptosis rate of renal tissue cells increased significantly in the experimental group (P＜0.01). Compared with the control group, the expression of ASPP2 in the experimental group increased at the early period and then decreased with the prolongation of injury time. The mRNA expression was consistent with the protein expression, and all reached the peak after 24 hours injury (P＜0.01). Immunohistochemistry showed that ASPP2 was mainly localized in the cytoplasm of renal tubular epithelial cells. Conclusions A single high-dose injection of CCl4 in the abdominal cavity can induce AKI in mice. The expression of ASPP2 is consistent with the degree of renal tissue damage. As the damage of renal tissue is aggravated, the expression of ASPP2 is gradually increased, which indicates that ASPP2 may be a damage factor.     

脑肠肽对脓毒症相关性肾损伤的保护作用研究

目的 探讨脑肠肽对内毒素所致大鼠脓毒症相关性肾损伤的影响.方法 健康雄性SD大鼠36只,随机分为3组：正常对照组,急性肾损伤（acute kidney injury,AKI）组,脑肠肽治疗组.采用内毒素静注制备脓毒症AKI模型,脑肠肽治疗组于造模前后30 min给予皮下注射脑肠肽（1.0mg/kg）,选择不同时间点（6 h、12 h、24 h）处死动物后留取血标本和肾组织,检测血清肌酐（SCr）、尿素氮（BUN）、血清肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）、观察肾组织的病理变化并检测肾脏组织中核因子κB的活化.结果 与同时间点比较,AKI组6h和12 h大鼠血清中TNF-α表达水平明显升高（P＜0.01）,24 h降至对照组水平（P＞0.05）,AKI组6h、12 h和24 h大鼠血清中BUN水平逐渐升高（P＜0.05）,24 h血清中SCr水平明显升高（P＜0.01）;AKI组24 h大鼠肾脏组织核因子κB p65核阳性率明显升高（P＜0.01）.脑肠肽治疗组对应时间点血清BUN和SCr水平较AKI组明显降低（P＜0.01）.TNF-α和核因子κB p65表达明显低于AKI组（P＜0.01）.病理显示脑肠肽治疗组大鼠肾损伤减轻,SCr、BUN、TNF-α、光镜检查均未见明显差异.结论 脑肠肽可通过抑制肾组织核因子κB的表达,下调TNF水平,对脓毒症相关性肾损伤发挥保护作用.     

脐带间充质干细胞调节炎性微环境干预急性肾损伤的效应研究

目的：观察移植外源性脐带间充质干细胞（UC-MSCs）对小鼠急性肾损伤（AKI）炎性微环境的影响及对肾组织的修复作用。方法：分离培养孕后期C57BL/6雌性小鼠的UC-MSCs。另取雌性C57BL/6小鼠60只,随机分为正常对照组、AKI组、AKI＋MSC移植组,每组20只。AKI组和AKI＋MSC组用微型动脉夹夹闭小鼠双侧肾蒂45 min复制AKI模型,并于模型制备成功时腹腔内注射生理盐水0.2 ml（AKI组）或1＆#215;106 UC-MSCs（AKI＋MSC组）。于第2天和第7天每组活杀10只小鼠,取眼球血及肾组织,检测血尿素氮（BUN）及血肌酐（SCr）水平;肾组织行HE染色,观察组织病理学变化;ELISA法检测肾组织匀浆中促炎因子IL-1β、TNF-α、干扰素-1（IFN-1）及抗炎因子碱性成纤维细胞生长因子（bFGF）、IL-10、B淋巴细胞瘤-2（Bcl-2）的水平。结果：2 d时AKI组小鼠肾小管上皮细胞肿胀,胞质空泡性变,BUN和Cr均显著高于正常对照组,提示造模成功后肾小管受损严重,7 d稍有减轻;AKI＋MSC移植组小鼠肾功能在2 d时较AKI组有所恢复,肾组织病理学变化明显减轻（P＜0.01）,7 d基本恢复正常。ELISA结果显示,与正常对照组比较, AKI组各时间点肾组织匀浆中促炎因子的水平均显著升高（P＜0.01或P＜0.05）,抗炎因子的水平均显著降低（P＜0.01或P＜0.05）,2 d较7 d更加明显。各时间点AKI＋MSC组抗炎因子的水平较AKI组明显升高,促炎因子的水平明显下降,但与正常对照组比较差异均有显著性（P＜0.01或P＜0.05）,7d较2 d改善更明显。结论：MSC可通过减轻AKI肾组织炎症反应,调节损伤肾脏组织微环境中的细胞因子水平发挥保护肾损害的修复作用。     

Role of OMA1 in lipopolysaccharide-induced acute kidney injury

Objective To investigate the role of OMA1 in acute kidney injury (AKI) induced by lipopolysaccharide (LPS). Methods OMA1 wild-type and knocked out mice (8 week old) were injected with 10 mg/kg body weight of LPS. The model was confirmed by testing mouse serum creatinine and blood urea nitrogen. The apoptosis in mouse kidney cortex was examined by TUNEL staining and cleaved caspase 3. In vitro, in humam kidney proximal tubular cells (HK2) were knocked down OMA1 by transfecting OMA1 shRNA, with the scramble shRNA being used as negative control of transfection. HK2 cells were cultured with 5 μg/ml of LPS for 24 hours to induce apoptosis. DAPI staining of cells and caspase-3 activity were applied to test apoptosis. The images of mitochondria in cells were obtained by transfection of mito-green plasmid and OMA1 shRNA. Western blotting was used to exam the OMA1 and Cytochrome C expressions. Results Compared with OMA1 KO mice, LPS induced more severe AKI of WT mice with higher Scr [(97.2±26.5) μmol/L vs (53.0±17.7) μmol/L, P＜0.05] and BUN [(43.3±13.7) mmol/L vs (29.7±7.7) mmol/L, P＜0.05]. Moreover, there were more apoptosis cells in kidney cortex in WT mice than in OMA1 KO mice [(75.4± 26.1)/mm2 vs (38.3± 14.4)/mm2, P＜0.05]. About 46% of OMA1 expressions in HK2 cells were inhibited by OMA1 shRNA transfection (P＜0.05). Further, OMA1 shRNA cells with LPS stimulation had decreased mitochondria fragmentation [(29.8±10.9)% vs (43.2±6.8)% , P＜0.05], Cytochrome C release [(37.0±12.3)% vs (76.0±26.2)%, P＜0.05], and cell apoptosis [(13.2±3.9)% vs (25.0±7.1)%, P＜0.05] as compared with control cells. Conclusion Knockdown of OMA1 alleviated septic AKI through inhibition of cell apoptosis, mitochondria fragmentation, and Cytochrome C release.     

半胱天冬蛋白酶-3在高迁移率族蛋白B1诱导小鼠巨噬细胞凋亡中的作用

目的 观察半胱天冬蛋白酶(Caspase)-3与核转录因子(NF)-KB在高迁移率族蛋白B1(HMGB1)诱导小鼠腹腔巨噬细胞凋亡中的作用.方法 分离培养小鼠腹腔巨噬细胞,随机分为对照组、HMCB1组与抑制剂组(HMGB1作用前1 h加入Caspase-3抑制剂Z-DEVD-FMK).采用Hoeehst33342染色观察凋亡的形态学变化、流式细胞术检测巨噬细胞凋亡百分率的变化;应用原位荧光染色及流式细胞术检测Caspase-3活性的变化;同时应用酶联免疫吸附试验检测NF-KB p65蛋白的变化.结果 HMGB1组巨噬细胞凋亡率为(38.21±4.85)%、抑制剂组为(16.47±3.91)%,均显著高于对照组(4.21±1.64)%(P＜0.01).经Hoechst33342染色,荧光显微镜观察HMGB1组凋亡细胞明显增多;原位荧光染色观察到HMGB1组Caspase-3荧光强度明显增强,Caspase-3阳性细胞百分率(29.74±4.55)%显著高于抑制剂组(3.02±1.91)%与对照组(7.19±2.46)%(P＜0.01).同时,HMGB1组、抑制剂组细胞核NF-KB活性较对照组显著降低(P＜0.05或P＜0.01).结论 Caspase-3和NF-KB是HMGB1诱导巨噬细胞凋亡的重要途径,Caspase-3抑制剂可部分阻断HMGB1诱导的巨噬细胞凋亡.     

Expression of tumor necrosis factor-α induced protein 8 like-1 in cisplatin-induced acute kidney injury models

Objective To investigate the expression and role of the tumor necrosis factor-α (TNF-α) induced protein 8 like-1 (TIPE1) in acute kidney injury (AKI) induced by cisplatin in animal model and cells. Methods Twelve male C57BL/6 mice aged 6-8 weeks were randomly divided into the control group and the model group. Mice in the model group received a single intraperitoneal injection of 20 mg/kg of cisplatin (20 mg/kg saline in the control group). All mice were euthanized after 5 days. Meanwhile, serum and kidney samples were collected. The levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were detected by biochemical kits. Renal histopathological changes in mice were observed by HE staining. The expression of TIPE1 in kidney was examined using immunohistochemistry. qRT-PCR was used for testing the relative expression of TIPE1 mRNA in mice kidney. Western blotting was used for testing TIPE1 and NGAL protein relative expression in mice kidney. Human kidney proximal tubular cells (HK-2) were stimulated with 20 μmol/L cisplatin for 0, 6, 12 and 24 h to establish cisplatin-induced AKI cell model. The expressions of TIPE1 mRNA and protein were detected by qRT-PCR and Western blotting in HK-2 cells. The expression of TIPE1 gene in HK-2 cells was silenced by lentivirus containing TIPE1 siRNA sequence. Then, TIPE1 stable knockout HK-2 cell strains were treated with 20 μmol/L of cisplatin for 24 hours. The protein expression of tubular damage marker neutrophil gelatinase-associated lipocalin (NGAL), microtubule-associated protein 1 light chain 3 (LC3) and Beclin1 in HK-2 cells were detected by Western blotting. Results Compared with the control group, the expressions of TIPE1 mRNA and protein were up-regulated and NGAL protein expression was increased significantly in renal tissue of the model group (all P＜0.05). The expressions of TIPE1 mRNA and protein were remarkably increased with the prolongation of cisplatin treatment in HK-2 cells (both P＜0.05). Compared with the scramble siRNA group, the protein expressions of NGAL, LC3-Ⅱ and Beclin1 were increased significantly in the TIPE1 siRNA group after lentivirus interfered with the expression of TIPE1 gene in HK-2 cells (all P＜0.05). Conclusions The mRNA and protein expressions of TIPE1 are increased in acute kidney injury models. Gene silencing of TIPE1 can promote the expressions of early renal tubular damage marker and autophagy-related proteins, which indicates the excessive autophagy aggravates renal tubular injury. It is suggested that TIPE1 may be involved in the pathogenesis of acute kidney injury.     

原发性肾病综合征患者中性粒细胞明胶酶相关脂质运载蛋白的变化及意义

目的 探讨原发性肾病综合征(PNS)患者中性粒细胞明胶酶相关脂质运载蛋白(NGAL)的变化以及与病理类型、肾小管间质损伤程度、临床指标的关系. 方法 40例PNS患者按有无急性肾小管坏死(ATN)分急性肾损伤(AKI)组及非AKI组,按病理类型分微小病变型肾病(MCD)组、系膜增生性肾小球肾炎(MsPGN)组、局灶性节段性肾小球硬化(FSGS)组、膜增生性肾小球肾炎(MPGN)组、膜性肾病(MN)组;20例健康体检者及20例因肾肿瘤做肾切除术但远离肿瘤部位的正常肾组织作正常对照.采用酶联免疫吸附试验(ELISA)法检测血清、尿液NGAL水平,免疫组织化学染色法观察肾组织NGAL表达.结果 (1) AKI组患者血、尿NGAL水平及肾组织NGAL表达显著高于非AKI组及对照组(P＜0.05).(2)MPGN组及FSGS组患者血、尿NGAL水平及肾组织NGAL表达显著高于其他病理类型组(P＜0.05).(3)在肾小管间质发展至重度病变之前,随着肾小管间质损伤程度的加重,血、尿NGAL水平及肾组织NGAL表达逐渐升高;在肾小管间质发展至重度病变时,血NGAL水平及肾组织NGAL表达下降(P＜0.05).(4)血、尿NGAL水平及肾组织NGAL表达与血肌酐呈正相关(r值分别为0.198、0.352、0.146,P值分别为0.048、0.000、0.028),与尿素氮呈正相关(r值分别为0.199、0.278、0.325,P值分别为0.043、0.000、0.019),与血白蛋白呈负相关(r值分别为-0.384、-0.318、-0.259,P值分别为0.028、0.024、0.020),与尿渗透浓度呈负相关(r值分别为-0.250、-0.256、-0.277,P值分别为0.012、0.027、0.002).结论 NGAL可作为预测PNS患者AKI的敏感指标,在一定程度下可用于评价肾小管间质病变程度及肾功能.     

胆管癌组织中高迁移率族蛋白B1与突变型p53的表达及临床意义

目的 探讨高迁移率族蛋白B1(HMGB1)和突变型p53(mutp53)在胆管癌组织中的表达及临床意义.方法 应用免疫组织化学方法检测47例胆管癌组织与25例正常胆管组织中HMGB1和mutp53的表达情况,并分析两者的表达与胆管癌临床病理特征之间的关系.结果 HMGB1与mutp53在胆管癌组织中的阳性表达率分别为78.72％ (37/47)、63.83％(30/47),而在正常胆管组织中的阳性表达率为12.00％(3/25)、4.00％(1/25),差异均有统计学意义(P＜0.01).HMGB1与mutp53在胆管癌组织中的阳性表达与患者的性别、年龄、胆红素水平、肿瘤直径大小及肿瘤所在部位均无相关性(P＞0.05);而与肿瘤的分化程度、淋巴结转移、神经侵犯及肿瘤的TNM分期密切相关,差异均有统计学意义(P＜0.05).HMGB1与mutp53在胆管癌组织中的表达呈正相关(r=0.574,P＜0.05).结论 HMGB1与mutp53在胆管癌组织中均呈高表达.HMGB1与mutp53在胆管癌的发生、发展、浸润及转移过程中起到重要作用.     

热射病对大鼠小肠上皮紧密连接occludin蛋白的影响

目的 观察热射病大鼠小肠上皮屏障通透性的改变,并通过观察肠上皮细胞紧密连接形态及肠上皮细胞紧密连接蛋白occludin蛋白表达的变化,初步探讨热射病对肠上皮屏障通透性的影响及机制.方法 将SD大鼠随机分为两组,每组10只(n=10),热射病组及常温对照组,制备热射病大鼠模型,动物麻醉后放入42 ℃恒温箱热暴露50 mi...     

Incidence and mortality of acute kidney injury in coronary care unit: a retrospective study from a single center

Objective To evaluate the incidence and mortality of acute kidney injury (AKI) in coronary care unit (CCU), and to identify the risk factors of the incidence of AKI and the mortality of CCU patients. Methods A total of 414 patients in CCU from January 1, 2014 to June 1, 2015 at Zhongnan Hospital of Wuhan University were enrolled. Based on the KDIGO-AKI criteria, these patients were classified into two groups: NAKI group (patients without AKI) and AKI group. Clinical characteristics and laboratory data of two groups were compared. The risk factors of the incidence of AKI and the mortality of CCU patients was analyzed by logistic regression, and then the receiver operating characteristic (ROC) curve was drawn to evaluate the predictive value of these risk factors. Results (1) Among 414 patients, 136(32.9%) patients fulfilled the criteria for AKI, and 14.0% patients in AKI stage 1, 10.9% in AKI stage 2 and 8.0% in AKI stage 3. (2) The total CCU mortality was 15.0%. Mortality of AKI patients in the CCU was 33.3%, higher than 6.1% in patients without AKI (OR=7.735, 95%CI 4.215-14.196, P＜0.001). The mortality worsened with increasing severity of AKI (22.4% for AKI stage 1 group, 37.8% for AKI stage 2 group, 45.4% for AKI stage 3 group). (3) Anemia (OR=8.274, 95%CI 4.363-15.689), history of chronic illness (OR=2.582, 95%CI 1.400-4.760), APACHEⅡ scores (OR=1.813, 95%CI 1.739-1.895), male (OR=3.666, 95%CI 1.860-7.226) were the independent risk factors for AKI, while the normal mean arterial pressure (MAP) (OR=0.292, 95%CI 0.153-0.556) and normal estimated glomerular filtration rate (eGFR) (OR=0.166, 95%CI 0.090-0.306) are the protective factors for AKI (all P＜0.05). (4) AKI was the most powerful independent factor associated with the mortality of CCU patients (OR=7.050, 95%CI 2.970-16.735, P＜0.001). Other independent risk factors for CCU mortality included history of chronic illness, ejection fraction and APACHEⅡ≥15 scores (all P＜0.05), while the normal MAP and normal eGFR were the protective factors (all P＜0.05). (5) For predicting AKI, eGFR displayed an excellent areas under the ROC curve (AUC=0.815, P＜0.001), and for CCU mortality, APACHEⅡ scores had the highest overall correctness of prediction (AUC=0.757 P＜0.001). Conclusions CCU patients have high morbidity of AKI, which is the most powerful independent factor associated with the increased CCU mortality. The eGFR is the best predictor for AKI, and then through the evaluation of eGFR for CCU patients, we can evaluate high-risk groups, make early interventions and then improve the prognosis of CCU patients.     

肾损伤分子-1、尿N-乙酰-β-D-氨基葡萄糖苷酶及尿微量白蛋白在重症感染中合并急性肾损伤的临床观察

目的 探讨尿肾损伤分子-1(Kim-1)、尿N-乙酰-β-D-氨基葡萄糖苷酶(N-acecyl-β-D-glucosaminidase,NAG)及尿微量白蛋白(mALB)在重症感染中合并急性肾损伤的敏感性及临床价值.方法 回顾分析60例在本院ICU住院的重症感染合并急性肾损伤(AKI)患者的尿Kim-1、尿NAG及尿m...     

维生素D受体在糖尿病肾病足细胞损伤及蛋白尿缓解中的作用

目的探讨维生素D受体(VDR)在糖尿病肾病(DKD)足细胞中的表达水平及在足细胞损伤及蛋白尿缓解中的作用。方法(1)本研究纳入了65例诊断患有2型糖尿病(伴或不伴蛋白尿)的患者,并纳入了25例年龄和性别相匹配的健康体检者为对照组。根据白蛋白/肌酐(ACR)的尿排泄比例对2型糖尿病患者进行分组,分别为无蛋白尿(ACR<30 mg/g,n=24)、微量白蛋白尿(ACR 30~300 mg/g,n=18)和临床蛋白尿(ACR>300 mg/g,n=23)。另选择25例经肾活检确诊的DKD患者作为DKD组。正常肾脏组织标本均取自泌尿外科同一时期肾脏肿瘤切除患者10例。将各组检测指标进行对比,同时采用实时定量PCR、ELISA法和免疫组化法检测VDR在各组患者的血液、尿液样本和肾脏组织中的表达情况,以及使用Pearson相关分析分析VDR与尿蛋白的相关性。(2)在2型糖尿病肾病小鼠模型中对上述结果进行验证,将遗传背景均为C57BLKs/J的雄性db/db小鼠及同窝出生的db/m小鼠,随机分为正常对照组(A组)、DKD对照组(B组)、DKD二甲基亚砜处理组(C组)、DKD帕立骨化醇(VDR激动剂)处理组(D组),C、D组连续腹腔注射处理8周,对照组不做任何处理。小鼠10周龄时开始连续干预8周,在小鼠22周龄(开始干预后12周)检测各组小鼠体重、血、尿生化指标对比;Western印迹法检测β⁃catenin、VDR的变化;免疫荧光观察足细胞标志蛋白podocin及足细胞损伤蛋白α⁃SMA的表达变化。结果(1)与正常健康对照组相比,无蛋白尿组、微量白蛋白尿组和临床蛋白尿组的糖尿病患者血浆中VDR的mRNA和蛋白水平均较低(均P<0.05);与无蛋白尿组的糖尿病患者相比,微量白蛋白尿组和临床蛋白尿组的糖尿病患者血浆中VDR的mRNA和蛋白水平均较低(均P<0.05)。(2)与正常健康对照组相比,无蛋白尿糖尿病组和DKD组患者血浆中VDR的mRNA和蛋白水平均较低(均P<0.05);与无蛋白尿糖尿病组患者相比,DKD组患者血浆中VDR的mRNA和蛋白水平亦较低(均P<0.05)。(3)免疫组化结果显示,DKD组肾组织中VDR的表达明显少于正常对照组。(4)DKD患者血浆中VDR mRNA相对水平与ACR呈负相关(r=-0.342,P<0.05)。(5)各组尿液上清液中VDR的水平与血浆中的水平呈相反趋势。(6)Western印迹结果显示,B组、C组肾小球足细胞β⁃catenin蛋白表达高于D组(均P<0.05),VDR蛋白的表达低于D组(均P<0.05);免疫荧光结果显示,B组、C组肾小球足细胞podocin的表达低于D组(均P<0.05),α⁃SMA的表达高于D组(均P<0.05)。结论VDR高表达缓解DKD足细胞损伤及蛋白尿。     

高迁移率蛋白族B1通过调控p53表达抑制T细胞增殖

目的:探讨高迁移率蛋白族B1 (HMGB1)对T细胞的增殖抑制作用及其分子机制.方法:将培养的Jurkat细胞分为对照组、HMGB1 (100 ng/ml) 12、24、48 h组,HMGB1组加入HMGB1培养相应时间;将细胞分为对照组、HMGB1 10、100、1 000 ng/ml组,予不同浓度HMGB1培养24 h;采用细胞计数试剂盒(CCK-8)法检测各组细胞增殖率,采用实时荧光定量-聚合酶链反应(RT-PCR)和Western blot法检测各组细胞中p53mRNA、磷酸化p53及p53总蛋白的表达水平.将载有p53 shRNA、p53 mRNA表达序列及空白质料的病毒转染入Jurkat细胞中,并选择100 ng/ml HMGB1刺激24 h,采用同样方法检测细胞增殖率.最后,将细胞分为正常组、HMGB1组、SB203580[p38丝裂原活化蛋白激酶(p38MAPK)抑制剂]+HMGB1组进行相应处理.收集各组细胞,RT-PCR和Western blot法检测p53 mRNA、磷酸化p53及p53总蛋白的表达水平.结果:HMGBl (100ng/ml)刺激细胞24、48 h后细胞增殖率降低,较正常组差异有显著性(P＜0.05);100、l 000 ng/ml HMGB1刺激细胞24 h后,增殖率明显下降,与正常组相比差异具有统计学意义(P＜0.05).HMGB1 (100 ng/ml)刺激细胞后,p53 mRNA、磷酸化及总蛋白表达水平在24、48 h逐步上升,且有明显统计学意义(P＜0.05);HMGB1刺激24 h,10、100 ng/ml HMGB1刺激组细胞p53 mRNA、p53磷酸化及总蛋白表达水平较正常组显著上升,但1 000 ng/mlHMGB1没有明显变化;在不同转染组中,予HMGB1刺激细胞,空载组增殖率降低,而p53 shRNA表达组细胞增殖趋于正常,p53 mRNA过表达组增殖率较空载组下降更为明显.Jurkat细胞在p38 MAPK阻断剂和HMGB1共同作用后,p53 mRNA、磷酸化及总蛋白表达水平比HMGB1刺激组明显下降(P＜0.05).结论:胞外HMGB1可通过诱导p38 MAPK信号通路激活p53蛋白,抑制T细胞增殖.     

先兆子痫大鼠循环、胎盘及肾脏局部血管紧张素Ⅱ及其1型受体的表达

目的 研究先兆子痫大鼠模型循环系统、胎盘及肾脏局部血管紧张素Ⅱ（AngⅡ）及其1型受体（AT1）的表达。 方法 采用一氧化氮合酶抑制剂亚硝基左旋精氨酸甲酯（L-NAME）制备大鼠先兆子痫模型，分别比较先兆子痫大鼠、正常妊娠大鼠、未孕对照组大鼠动脉收缩压（SBP）、尿蛋白量(24 h)、肝肾功能，并对各组大鼠肾组织进行光镜检查。ELISA法和放射性免疫法分别测定各组大鼠血浆及肾脏局部匀浆液AngⅡ水平。Western印迹法检测大鼠胎盘局部AT1表达。免疫组化法及Western印迹法测定大鼠肾脏局部AT1的表达。 结果 在先兆子痫大鼠中，SBP及尿蛋白量(24 h)均较未孕对照组显著升高（P < 0.05）。先兆子痫大鼠血浆AngⅡ显著高于正常妊娠组[（0.706±0.086） ng/L比(0.540±0.085) ng/L，P < 0.05]；胎盘局部AT1表达比正常妊娠组升高46％（P < 0.05）；肾脏局部AngⅡ明显低于正常妊娠组[(65.543±40.634) ng/g比(165.543±33.078) ng/g，P < 0.05]；肾脏AT1表达减少，仅为正常妊娠组及未孕对照组的33％及59％（P < 0.05）。 结论 先兆子痫中，胎盘局部RAS表达增强，循环AngⅡ表达增高，肾脏局部RAS表达下调。     

高迁移率族蛋白1对狼疮肾炎小鼠肾小球细胞增殖的影响

目的 通过敲低肾组织高迁移率族蛋白1(HMGB1)表达,探讨其对改善狼疮肾炎小鼠肾功能,降低肾小球细胞增殖水平的影响.方法 MRL/Faslpr鼠(n=24)被随机分为模型组、shHMGB1组和空质粒组;选取周龄、体质量相匹配的MRL/MpJ鼠为健康对照组.shHMGB1组和空质粒组采用电穿孔转染技术分别转染shHMGB1质粒和空质粒,模型组和健康对照组仅转染生理盐水.用全自动生化分析仪检测小鼠血清尿素氮和肌酐水平,测定尿蛋白浓度并计算24 h尿蛋白量(UP).HE染色观察肾组织的形态学表现;免疫荧光和Western印迹法检测小鼠肾小球中HMGB1和增殖细胞核抗原(PCNA)的表达;实时定量PCR法检测小鼠肾小球HMGB1和PCNA mRNA表达变化.结果 (1)与健康对照组相比,模型组小鼠肾小球HMGB1 mRNA和蛋白的表达升高(均P＜0.05);与模型组相比,shHMGB1组小鼠肾小球中HMGB1 mRNA和蛋白的表达降低(均P＜0.05).(2)与模型组相比,shHMGB1组小鼠尿蛋白减少(P＜0.05).(3)免疫荧光和Western印迹结果显示,与健康对照组相比,模型组小鼠肾小球中PCNA mRNA和蛋白表达升高(P＜0.05).与模型组相比,shHMGB1组肾小球中PCNA表达降低(P＜0.05).结论 敲低肾组织HMGB1表达可改善狼疮肾炎小鼠的肾功能,降低肾小球细胞的增殖水平.     

Renoprotective effect of transforming growth factor beta activator kinase 1 inhibitor in diabetic db/db mice and its mechanism

Objective To investigate the renoprotective effect of transforming growth factor beta activator kinase 1 (TAK1) inhibitor 5Z-7-oxozeaenol (OZ) in diabetic db/db mice and the mechanism. Methods Twenty-four male db/db mice were randomly divided into two groups: db/db mice (db/db, n=12) and db/db mice with 5Z-7-oxozeaenol treatment (db/db+OZ, n=12). Another group of wild type mice (n=12) was held as the control group. OZ 2 mg/kg was administrated by intraperitoneal injection every other day. At week 8 and 12 after 5Z-7-oxozeaenol treatment, blood glucose (BG), body weight (BW), kidney weight (KW) and urinary albumin excretion rate (UAER) were evaluated. Kidney pathological lesions were detected by light and electron microscopy. NF-κB p65, monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) were detected by immunohistochemistry. Western blotting was used to detect p-TAK1, TAB1, p-p38MAPK and IL-1β expression, while ICAM-1 and MCP-1 mRNA levels were evaluated by real-time PCR. Results Compared with control group, the levels of BG, BW, KW and UAER were higher (P＜0.01) in db/db mice group, while BW, KW and UAER levels were significantly decreased in db/db+OZ group compared with that in db/db mice group (P＜0.05). In week 8 and 12 db/db mice, glomerular volume and extracellular matrix were increased, while pathological lesions in kidney tissue were positively improved by TAK1 inhibitor. Immunohistochemistry showed that NF-κB p65, MCP-1 and TNF-α expression levels were apparently increased in db/db mice group compared with that in control group (P＜0.05) and were significantly inhibited by TAK1 inhibitor (P＜0.05). Western blotting showed that p-TAK1, TAB1, p-p38MAPK and IL-1β expression levels were higher in db/db mice group than that in control group (P＜0.05) and lower in db/db+OZ group than that in db/db mice group (P＜0.05). Moreover, real-time PCR showed that the expressions of ICAM-1 and MCP-1 mRNA were higher in db/db mice group than that in control group and lower in db/db+OZ group than that in db/db mice group (P＜0.05). Conclusions TAK1 Inhibitor can down-regulate MAPK and NF-κB pathway to restrain the reaction of inflammation and alleviate kidney injury in diabetic db/db mice.     

Early interleukin 6 production by leukocytes during ischemic acute kidney injury is regulated by TLR4

Although leukocytes infiltrate the kidney during ischemic acute kidney injury (AKI) and release interleukin 6 (IL6), their mechanism of activation is unknown. Here, we tested whether Toll-like receptor 4 (TLR4) on leukocytes mediated this activation by interacting with high-mobility group protein B1 (HMGB1) released by renal cells as a consequence of ischemic kidney injury. We constructed radiation-induced bone marrow chimeras using C3H/HeJ and C57BL/10ScNJ strains of TLR4 (-/-) mice and their respective TLR4 (+/+) wild-type counterparts and studied them at 4?h after an ischemic insult. Leukocytes adopted from TLR4 (+/+) mice infiltrated the kidneys of TLR4 (-/-) mice, and TLR4 (-/-) leukocytes infiltrated the kidneys of TLR4 (+/+) mice but caused little functional renal impairment in each case. Maximal ischemic AKI required both radiosensitive leukocytes and radioresistant renal parenchymal and endothelial cells from TLR4 (+/+) mice. Only TLR4 (+/+) leukocytes produced IL6 in vivo and in response to HMGB1 in vitro. Thus, following infiltration of the injured kidney, leukocytes produce IL6 when their TLR4 receptors interact with HMGB1 released by injured renal cells. This underscores the importance of TLR4 in the pathogenesis of ischemic AKI.     

Mediation of inflammatory activation of renal tubular epithelial cells by high mobility group protein box 1 interacting with Toll-like receptor 4

Objective To observe functional changes of renal tubular epithelial cells stimulated by high mobility group protein box 1 (HMGB1) and associated mechanism. Methods Renal tubular epithelial cells (NRK52E) were divided into control group, HMGB1 group and HMGB1+lipopolysaccharide from Rhodobactersphaeroides (LPS RS) group. Toll-like receptor 4 (TLR4) expression was detected by immunofluorescence and Western blotting. Apoptosis rate and cell cycle arrest were identified with flow cytometry. The activation of MAPK signaling pathway and NF-κB were detected by Western blotting. The IL-1, IL-6 and tissue inhibitor of metalloproteinases 2 (TIMP2) mRNA levels were measured by real-time PCR. The secretion levels of IL-1, IL-6 and TIMP2 were measured by protein chips assay. Results TLR4 was expressed by NRK52E cells. Compared with the control group, there were increased cell cycle G1 arrest, MAPK signaling pathway and NF-κB activation in HMGB1 group. Furthermore, IL-1, IL-6 and TIMP2 mRNA levels were increased and IL-1, IL-6 and TIMP2 were secreted by NRK52E when stimulated with HMGB1 (all P＜0.05). However, effects mediated by HMGB1 stimulation could be inhibited by LPS RS (all P＜0.05). Conclusions Inflammatory activation of NRK52E cells can be mediated by the interaction of HMGB1 and TLR4.