Intermedin对肾脏缺血再灌注损伤保护作用的实验研究
摘要
垂体中叶素Intermedin(IMD)是新发现的内源性心血管、肾脏保护因子,属降钙素基因相关肽(calcitonin gene related peptide, CGRP)超家族成员,对其在肾脏的保护机制尚不清楚。本研究采用分子生物学及细胞生物学手段,对IMD在肾脏缺血再灌注损伤(IRI)中的病理生理意义及其机制进行探讨。分为以下两个部分:
一、大鼠肾脏缺血再灌注损伤模型中IMD及其受体的变化
目的:制备大鼠肾脏IRI模型,观察IMD及其受体系统的变化,初步探讨IMD在肾脏IRI中的作用。
方法:健康雄性Wistar大鼠18只,随机分为假手术组、缺血组和IRI组。双侧夹闭肾动脉45min,再灌注12h制作IRI模型。检测血清尿素氮(BUN)、肌酐(SCr)浓度,并半定量分析肾脏病理组织学变化判断模型成功与否。放射免疫分析法测定血浆、肾组织IMD及血浆肾上腺髓质素(ADM)含量。半定量反转录-聚合酶链反应(RT-PCR)法检测肾组织IMD、ADM及其受体系统CRLR、RAMP1、2、3 mRNA表达;Western blotting方法半定量分析肾组织IMD及其受体CRLR的蛋白表达。
结果:⑴与假手术组相比,IRI组大鼠BUN和SCr明显升高(P<0.05);病理组织学显示IRI组大鼠肾小管上皮细胞空泡状变性、刷状缘坏死脱落等,而肾小球无明显改变,半定量评分显示该组与假手术组和缺血组之间存在统计学差异(分别为P<0.05,P<0.01),符合急性肾损伤病理改变,造模成功。⑵大鼠血浆、肾组织IMD含量,IRI组较假手术组升高53.76%、40.01%(P<0.05);较缺血组升高58.34%、42.97%(P<0.05);血浆ADM含量,IRI组较假手术组升高143.22%(P<0.01),较缺血组升高101.96%(P<0.01)。⑶与假手术组相比,IRI组肾组织IMD、ADM、CRLR、RAMP1、RAMP2、RAMP3 mRNA相对含量分别上调了22.1%(P<0.05)、41.1%(P<0.01)、32.8%(P<0.01)、33.7%(P<0.01)、35.9%(P<0.05)、29.4%(P<0.01);IMD和CRLR蛋白表达明显增高,分别增加了80.9%,68.4%(P<0.001)。
结论:肾脏IRI模型中血浆和肾组织IMD水平显著增加,同时肾组织IMD及受体系统CRLR/RAMPs的mRNA表达和肾组织IMD、CRLR的蛋白表达均出现与血浆和肾组织IMD水平变化趋势一致的增加,提示IMD参与了肾脏IRI的病理生理过程,可能在保护肾脏方面有重要意义。
二、IMD对大鼠肾小管上皮细胞缺氧复氧模型的影响及机制研究
目的:以肾小管上皮细胞(NRK-52E)缺氧复氧(H/R)模型模拟在体IRI,通过转染IMD真核表达质粒,探讨IMD细胞保护的分子机制。
方法:⑴利用三气培养箱调整氮气压力形成缺氧条件,在缺氧1h,复氧1.5h后检测NRK-52E活细胞计数、细胞存活率和培养液上清乳酸脱氢酶(LDH)含量判断H/R模型成功与否。⑵人工合成IMD目的片段,与pMD19-T Simple载体连接形成克隆载体,再通过双酶切定向克隆技术将其亚克隆至pIRES2-EGFP,构建IMD真核表达载体,命名为pIRES2-EGFP/IMD,测序鉴定。⑶利用Fugene HD转染试剂,将pIRES2-EGFP/IMD表达质粒转染至NRK-52E细胞内,以倒置荧光显微镜和流式细胞仪检测转染效率,选定最优转染条件;并以RT-PCR和Western blotting方法观察其对细胞IMD mRNA和蛋白表达的影响;以G418 300μg/ml加入10%胎牛血清的DMEM/F12完全培养基对细胞进行筛选约2周,获得稳定表达IMD的阳性克隆NRK-52E,Western blotting鉴定筛选后IMD蛋白的表达。⑷对NRK-52E进行H/R实验,分为对照组和6个模型组包括单纯H/R组、空质粒组、IMD质粒组、IMD+PKA抑制剂(H-89)组、环磷酸腺苷(cAMP)类似物(8-Br-cAMP)组和NADPH氧化酶抑制剂(DPI)组,分别检测细胞培养液上清中LDH、cAMP含量,细胞NADPH氧化酶活性、超氧化物歧化酶(SOD)、丙二醛(MDA)和活性氧(ROS)水平,观察IMD对H/R时氧化损伤的影响及分子机制。
结果:⑴经缺氧1h,复氧1.5h后,NRK-52E细胞计数量降低,存活率下降,细胞培养液上清中LDH含量显著增加(P<0.05),造模成功。⑵酶切和测序鉴定表明,pMD19-T Simple/IMD克隆质粒和pIRES2-EGFP/IMD真核表达载体构建成功。⑶荧光显微镜和流式细胞仪检测结果显示以质粒3μg: FuGENE HD 12μl配比的转染复合体在48h的转染效率最高,转染效率最高可达71.34±6.24%(P<0.01);半定量RT-PCR和Western blotting结果显示,转重组质粒组IMD mRNA表达量较未转染组增高(P<0.01),蛋白表达也显著增高(P<0.001)。G418筛选后转染阳性的细胞于第3天起逐渐出现克隆样增殖,第5天起未转染的细胞出现批量死亡,以后阳性细胞克隆逐渐增加,至第14天可见融合成片状的阳性克隆细胞,约占总细胞的60~70%。Western blotting结果显示,筛选后IMD蛋白的表达呈现高表达,说明细胞得到稳定表达。⑷给予各种干预因素后,各检测指标的变化如下:①LHD含量:与对照组相比,各模型组LDH显著上升(P﹤0.01);与H/R组相比,IMD质粒组与8-Br-cAMP组LDH上升幅度较小(P﹤0.05)。②cAMP含量:与对照组相比,各模型组cAMP含量明显增加,其中IMD质粒+H-89组和8-Br-cAMP组升高最为显著(P﹤0.01);与H/R组相比,IMD质粒+H-89组和8-Br-cAMP组的cAMP含量上升幅度最显著(P﹤0.01),IMD质粒组也有所上升,DPI组却有所下降(P﹤0.05)。③NADPH氧化酶含量:除DPI组外各模型组NADPH氧化酶含量均明显升高(P﹤0.01),其中8-Br-cAMP组升高幅度最小(P﹤0.05);与H/R组比较,8-Br-cAMP组和DPI组的NADPH氧化酶含量分别下降了33.92%(P﹤0.05)、52.54%(P﹤0.01)。④MDA含量:与对照组相比,模型组中H/R组(P﹤0.01)、空质粒组(P﹤0.01)、IMD质粒+ H-89组(P﹤0.05)和DPI组(P﹤0.05)的MDA含量增加;与H/R组比较,IMD质粒组、IMD质粒+ H-89组、8-Br-cAMP组和DPI组的MDA均有所下降(P﹤0.05)。⑤ROS含量:与对照组相比,模型组中H/R组、空质粒组、IMD质粒+ H-89组的ROS含量增加(P﹤0.05);与H/R组比较,IMD质粒组的ROS含量有所下降(P﹤0.05)。⑥SOD含量:模型组中IMD质粒组、IMD质粒+ H-89组和DPI组的SOD含量有所升高(P﹤0.05),其余各组与对照组和H/R组相比均无明显差别。
结论:在肾小管上皮细胞H/R模型中,IMD可通过cAMP/PKA途径增加cAMP水平,抑制NADPH氧化酶活性,减少ROS产生,同时上调SOD,减轻细胞氧化损伤,参与氧化与抗氧化平衡的调节,作为重要的内源性抗氧化剂实现其细胞保护作用。
Being a newly-found endogenous cardio-renal-protective substance, Intermedin (IMD) belongs to the calcitonin gene-related peptide (CGRP) superfamily; its renal-protection mechanism is still unknown. Applying the methods of both cytobiology and molecular biology, this study is designed to explore the physiological and pathological significance of IMD in renal ischemia-reperfusion injury(IRI). This study includes two parts:
1. The changes of IMD and its receptors in rat renal ischemia reperfusion injury
Objective The rat models of renal ischemia reperfusion injury(IRI) were established to observe the changes of IMD and its receptors after IRI, and explore the functions of IMD in renal IRI. Methods Eighteen male Wistar rats were randomly divided into sham-operated,
ischemia and IRI groups. IRI rat models were made by clamping both renal arteries for 45min and reperfusing for 12 hours. IRI models were evaluated through measuring blood urea nitrogen(BUN) and serum creatinine(SCr) concentration, and through semi-quantitated anaylsis of renal histological changes. The contents of IMD and ADM in plasma and renal tissue were assessed by radioimmunoassay (RIA). The mRNA expressions of IMD, ADM, CRLR and RAMPs in the kidneys were determined by semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR). The changes of protein expressions of IMD and CRLR in the kidneys were detected by Western blotting.
Results⑴Compared with the sham ones, BUN and SCr in IRI rats increased significantly(P<0.05). Kidneys pathologic changes of IRI rat models were significant, as shown in tubule vacuolar degeneration or necrosis, loss of brush border; while there was little change in glomerulus. Semi-quantitated analysis suggests there was significant difference between IRI groups and the other two groups. (the sham-operated groups, P<0.05 and the ischemia groups, P<0.01). The results go with the changes of acute renal injury, so models are successfully established.⑵Compared with sham-operated and ischemia groups, the IMD contents of serum and renal in IRI group increased by 53.76%, 40.01% and 58.34%, 42.97% (P<0.05), and serum ADM showed a marked increase by 143.22%, 101.96% (P<0.05).⑶Compared with sham-operated group, the mRNA expressions of IMD, ADM, CRLR, RAMP1, RAMP2, RAMP3 in kidneys of IRI group were all up-regulated significantly by 22.1%(P<0.05, 41.1%(P<0.01), 32.8%(P<0.01), 33.7%(P<0.01), 35.9%(P<0.05), 29.4%(P<0.01), and the protein expressions of IMD and CRLR in kidneys increased significantly by 80.9%, 68.4%(P<0.001).
Conclusion In IRI group, levels of IMD in blood plasma and kidney increased significantly; and consistently there appeared marked increases of the mRNA expressions of IMD and of its receptors, and the protein expressions of IMD and CRLR in kidneys. These results suggest that IMD may have participated in the process of renal IRI, and may have important physiological and pathological significance in terms of kidney protection.
2. The influence of IMD exerted on the hypoxia/reoxygenation model of rat renal tubular epithelial cells and its mechanism
Objective The hypoxia/reoxygenation(H/R) injury models of rat renal tubular epithelial cells (NRK-52E), which were transfected the eukaryotic expression vector of rat IMD, were established to simulate IRI in vivo, to investigate the molecule mechanism of IMD cytoprotection.
Methods⑴To establish the H/R injury model of NRK-52E by regulating the pressure of N2 in incubator to hypoxia condition, the cells were cultured with hypoxia for 1h and then with reoxygenation for 1.5h. The models were evaluated by detecting living cell count, cell viability and the activity of lactate dehydrogenase (LDH) in the culture medium.⑵The full length IMD cDNA, which was synthesized artificially, was connected with pMD19-T Simple vector to form a cloned vector. This cloned vector was sub-cloned into pIRES2-EGFP to construct an eukaryotic expression vector, which is named pIRES2-EGFP/IMD. Restriction enzyme digestion and sequencing analysis were adopted to test the recombinant plasmids.⑶NRK-52E cells were transfected by transfection complex comprising optimal proportion of pIRES2-EGFP/IMD plasmid and Fugene HD reagents. Transfection efficiency was tested by observation for EGFP made by fluorescent microscope and flow cytometery (FCM) after 48h. Total RNA and protein was extracted from EGFP positive NRK-52E cells. IMD mRNA and protein expression level was evaluated by RT-PCR and Western-blotting. The cell groups with high transfection efficiency were screened for two weeks or so by incubating with medium containing G418(300μg/ml), and the positive cloning NRK-52E cells of stable expression IMD obtained. The expression of IMD protein in IMD plasmid group after screening was evaluated by Western-blotting.⑷The NRK-52E cells were divided into 7 groups. One of them was control group; the other six model groups were exposed to H/R condition, following the intervention of single H/R, primitive vector, highly expressed IMD vector, NADPH oxidase inhibitor(DPI), cyclic adenosine monophosphate(cAMP) analogue (8-Br-cAMP), PKA inhibitor(H-89), respectively. The contents of LDH and cAMP in the culture medium and the levels of NADPH oxidase, superoxide dismutase(SOD), malondialdehyde(MDA) and reactive oxygen species(ROS) in cells were detected to observe the influence of IMD on H/R injury and its molecule mechanism.
Results⑴After hypoxia 1h and reoxygenation 1.5h, cell count and cell viability decreased significantly and the activity of LDH increased significantly (P<0.05); the model was established successfully.⑵Restriction enzyme digestion and sequencing analysis showed that pMD19-T Simple/IMD cloning vector and pIRES2-EGFP/IMD eukaryotic expression vector had been constructed successfully.⑶NRK-52E cells was transfected with the optimal transfection proportion of pIRES2-EGFP/IMD to Fugene HD reagent as 3μg: 12μl, transfection efficiency evaluated by fluorescence microscope and FCM showed that the transfection efficiency increased with the prolongation of time and reached its peak 71.34±6.24% at 48h(P<0.01). The result of RT-PCR and Western blotting showed the expression levels of IMD mRNA and protein in IMD plasmid group were increased significantly (P<0.01 and P<0.001, respectively). The positive transfected cells after G418 screening were found clone-like proliferation on the third day, and the untransfected cells died in large bulk on the fifth day. Then the positive transfected cells increased gradually; and the positive cloning cells blending into slices were found on the fourteenth day, which occupied 60-70% of the total cell number. The result of Western blotting showed the expression of IMD protein in IMD plasmid group after screening increased significantly, which indicate the IMD expression of the cell is stable.⑷In NRK-52E cells following the seven treatments, the changes of detected index were shown:①Contrasted with the control group, the level of LDH of all model groups increased significantly (P<0.01); but the extent of increase in IMD plasmid group and 8-Br-cAMP group were smaller than that in H/R group(P﹤0.05).②Contrasted with the control group, the level of cAMP of all model groups also increased significantly(P<0.01), and IMD plasmid + H-89 group and 8-Br-cAMP group were exceptionally noticeable; when compared with H/R group, the two groups were the most remarkably increased groups(P﹤0.01), IMD plasmid group increased mildly and DPI group decreased(P﹤0.05).③Contrasted with the control group, the activity of NADPH oxidase of all model groups increased significantly except DPI group(P<0.01), but the extent of increase in 8-Br-cAMP group was the smallest. Compared with H/R group, the levels of NADPH oxidase of 8-Br-cAMP group and DPI group decreased by 33.92%(P﹤0.05) and 52.54%(P﹤0.01), respectively.④The content of MDA increased in H/R group (P﹤0.01), primitive plasmid group (P﹤0.01), IMD plasmid + H-89 group (P﹤0.05) and DPI group (P﹤0.01), but decreased in IMD plasmid group, IMD plasmid + H-89 group, 8-Br-cAMP group and DPI group(P﹤0.05) compared with H/R group.⑤The content of ROS increased in H/R group, primitive plasmid group, IMD plasmid + H-89 group (P﹤0.05), but decreased in IMD plasmid group compared with H/R group(P﹤0.05).⑥The content of SOD increased in IMD plasmid group, IMD plasmid + H-89 group and DPI group(P﹤0.05), but it changed little in the other four groups.
Conclusion In H/R model of NRK-52E, IMD could increase the level of cAMP via cAMP/PKA access, inhibit the activity of NADPH oxidase, decrease the production of ROS, upgrade SOD, alleviate the cell oxidative damage, regulate the oxidative and anti-oxidative balance. All these suggest that IMD, as an endogenous antioxidant, plays a protective role in cell protection during H/R-induced oxidative injury.
一、大鼠肾脏缺血再灌注损伤模型中IMD及其受体的变化
目的:制备大鼠肾脏IRI模型,观察IMD及其受体系统的变化,初步探讨IMD在肾脏IRI中的作用。
方法:健康雄性Wistar大鼠18只,随机分为假手术组、缺血组和IRI组。双侧夹闭肾动脉45min,再灌注12h制作IRI模型。检测血清尿素氮(BUN)、肌酐(SCr)浓度,并半定量分析肾脏病理组织学变化判断模型成功与否。放射免疫分析法测定血浆、肾组织IMD及血浆肾上腺髓质素(ADM)含量。半定量反转录-聚合酶链反应(RT-PCR)法检测肾组织IMD、ADM及其受体系统CRLR、RAMP1、2、3 mRNA表达;Western blotting方法半定量分析肾组织IMD及其受体CRLR的蛋白表达。
结果:⑴与假手术组相比,IRI组大鼠BUN和SCr明显升高(P<0.05);病理组织学显示IRI组大鼠肾小管上皮细胞空泡状变性、刷状缘坏死脱落等,而肾小球无明显改变,半定量评分显示该组与假手术组和缺血组之间存在统计学差异(分别为P<0.05,P<0.01),符合急性肾损伤病理改变,造模成功。⑵大鼠血浆、肾组织IMD含量,IRI组较假手术组升高53.76%、40.01%(P<0.05);较缺血组升高58.34%、42.97%(P<0.05);血浆ADM含量,IRI组较假手术组升高143.22%(P<0.01),较缺血组升高101.96%(P<0.01)。⑶与假手术组相比,IRI组肾组织IMD、ADM、CRLR、RAMP1、RAMP2、RAMP3 mRNA相对含量分别上调了22.1%(P<0.05)、41.1%(P<0.01)、32.8%(P<0.01)、33.7%(P<0.01)、35.9%(P<0.05)、29.4%(P<0.01);IMD和CRLR蛋白表达明显增高,分别增加了80.9%,68.4%(P<0.001)。
结论:肾脏IRI模型中血浆和肾组织IMD水平显著增加,同时肾组织IMD及受体系统CRLR/RAMPs的mRNA表达和肾组织IMD、CRLR的蛋白表达均出现与血浆和肾组织IMD水平变化趋势一致的增加,提示IMD参与了肾脏IRI的病理生理过程,可能在保护肾脏方面有重要意义。
二、IMD对大鼠肾小管上皮细胞缺氧复氧模型的影响及机制研究
目的:以肾小管上皮细胞(NRK-52E)缺氧复氧(H/R)模型模拟在体IRI,通过转染IMD真核表达质粒,探讨IMD细胞保护的分子机制。
方法:⑴利用三气培养箱调整氮气压力形成缺氧条件,在缺氧1h,复氧1.5h后检测NRK-52E活细胞计数、细胞存活率和培养液上清乳酸脱氢酶(LDH)含量判断H/R模型成功与否。⑵人工合成IMD目的片段,与pMD19-T Simple载体连接形成克隆载体,再通过双酶切定向克隆技术将其亚克隆至pIRES2-EGFP,构建IMD真核表达载体,命名为pIRES2-EGFP/IMD,测序鉴定。⑶利用Fugene HD转染试剂,将pIRES2-EGFP/IMD表达质粒转染至NRK-52E细胞内,以倒置荧光显微镜和流式细胞仪检测转染效率,选定最优转染条件;并以RT-PCR和Western blotting方法观察其对细胞IMD mRNA和蛋白表达的影响;以G418 300μg/ml加入10%胎牛血清的DMEM/F12完全培养基对细胞进行筛选约2周,获得稳定表达IMD的阳性克隆NRK-52E,Western blotting鉴定筛选后IMD蛋白的表达。⑷对NRK-52E进行H/R实验,分为对照组和6个模型组包括单纯H/R组、空质粒组、IMD质粒组、IMD+PKA抑制剂(H-89)组、环磷酸腺苷(cAMP)类似物(8-Br-cAMP)组和NADPH氧化酶抑制剂(DPI)组,分别检测细胞培养液上清中LDH、cAMP含量,细胞NADPH氧化酶活性、超氧化物歧化酶(SOD)、丙二醛(MDA)和活性氧(ROS)水平,观察IMD对H/R时氧化损伤的影响及分子机制。
结果:⑴经缺氧1h,复氧1.5h后,NRK-52E细胞计数量降低,存活率下降,细胞培养液上清中LDH含量显著增加(P<0.05),造模成功。⑵酶切和测序鉴定表明,pMD19-T Simple/IMD克隆质粒和pIRES2-EGFP/IMD真核表达载体构建成功。⑶荧光显微镜和流式细胞仪检测结果显示以质粒3μg: FuGENE HD 12μl配比的转染复合体在48h的转染效率最高,转染效率最高可达71.34±6.24%(P<0.01);半定量RT-PCR和Western blotting结果显示,转重组质粒组IMD mRNA表达量较未转染组增高(P<0.01),蛋白表达也显著增高(P<0.001)。G418筛选后转染阳性的细胞于第3天起逐渐出现克隆样增殖,第5天起未转染的细胞出现批量死亡,以后阳性细胞克隆逐渐增加,至第14天可见融合成片状的阳性克隆细胞,约占总细胞的60~70%。Western blotting结果显示,筛选后IMD蛋白的表达呈现高表达,说明细胞得到稳定表达。⑷给予各种干预因素后,各检测指标的变化如下:①LHD含量:与对照组相比,各模型组LDH显著上升(P﹤0.01);与H/R组相比,IMD质粒组与8-Br-cAMP组LDH上升幅度较小(P﹤0.05)。②cAMP含量:与对照组相比,各模型组cAMP含量明显增加,其中IMD质粒+H-89组和8-Br-cAMP组升高最为显著(P﹤0.01);与H/R组相比,IMD质粒+H-89组和8-Br-cAMP组的cAMP含量上升幅度最显著(P﹤0.01),IMD质粒组也有所上升,DPI组却有所下降(P﹤0.05)。③NADPH氧化酶含量:除DPI组外各模型组NADPH氧化酶含量均明显升高(P﹤0.01),其中8-Br-cAMP组升高幅度最小(P﹤0.05);与H/R组比较,8-Br-cAMP组和DPI组的NADPH氧化酶含量分别下降了33.92%(P﹤0.05)、52.54%(P﹤0.01)。④MDA含量:与对照组相比,模型组中H/R组(P﹤0.01)、空质粒组(P﹤0.01)、IMD质粒+ H-89组(P﹤0.05)和DPI组(P﹤0.05)的MDA含量增加;与H/R组比较,IMD质粒组、IMD质粒+ H-89组、8-Br-cAMP组和DPI组的MDA均有所下降(P﹤0.05)。⑤ROS含量:与对照组相比,模型组中H/R组、空质粒组、IMD质粒+ H-89组的ROS含量增加(P﹤0.05);与H/R组比较,IMD质粒组的ROS含量有所下降(P﹤0.05)。⑥SOD含量:模型组中IMD质粒组、IMD质粒+ H-89组和DPI组的SOD含量有所升高(P﹤0.05),其余各组与对照组和H/R组相比均无明显差别。
结论:在肾小管上皮细胞H/R模型中,IMD可通过cAMP/PKA途径增加cAMP水平,抑制NADPH氧化酶活性,减少ROS产生,同时上调SOD,减轻细胞氧化损伤,参与氧化与抗氧化平衡的调节,作为重要的内源性抗氧化剂实现其细胞保护作用。
Being a newly-found endogenous cardio-renal-protective substance, Intermedin (IMD) belongs to the calcitonin gene-related peptide (CGRP) superfamily; its renal-protection mechanism is still unknown. Applying the methods of both cytobiology and molecular biology, this study is designed to explore the physiological and pathological significance of IMD in renal ischemia-reperfusion injury(IRI). This study includes two parts:
1. The changes of IMD and its receptors in rat renal ischemia reperfusion injury
Objective The rat models of renal ischemia reperfusion injury(IRI) were established to observe the changes of IMD and its receptors after IRI, and explore the functions of IMD in renal IRI. Methods Eighteen male Wistar rats were randomly divided into sham-operated,
ischemia and IRI groups. IRI rat models were made by clamping both renal arteries for 45min and reperfusing for 12 hours. IRI models were evaluated through measuring blood urea nitrogen(BUN) and serum creatinine(SCr) concentration, and through semi-quantitated anaylsis of renal histological changes. The contents of IMD and ADM in plasma and renal tissue were assessed by radioimmunoassay (RIA). The mRNA expressions of IMD, ADM, CRLR and RAMPs in the kidneys were determined by semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR). The changes of protein expressions of IMD and CRLR in the kidneys were detected by Western blotting.
Results⑴Compared with the sham ones, BUN and SCr in IRI rats increased significantly(P<0.05). Kidneys pathologic changes of IRI rat models were significant, as shown in tubule vacuolar degeneration or necrosis, loss of brush border; while there was little change in glomerulus. Semi-quantitated analysis suggests there was significant difference between IRI groups and the other two groups. (the sham-operated groups, P<0.05 and the ischemia groups, P<0.01). The results go with the changes of acute renal injury, so models are successfully established.⑵Compared with sham-operated and ischemia groups, the IMD contents of serum and renal in IRI group increased by 53.76%, 40.01% and 58.34%, 42.97% (P<0.05), and serum ADM showed a marked increase by 143.22%, 101.96% (P<0.05).⑶Compared with sham-operated group, the mRNA expressions of IMD, ADM, CRLR, RAMP1, RAMP2, RAMP3 in kidneys of IRI group were all up-regulated significantly by 22.1%(P<0.05, 41.1%(P<0.01), 32.8%(P<0.01), 33.7%(P<0.01), 35.9%(P<0.05), 29.4%(P<0.01), and the protein expressions of IMD and CRLR in kidneys increased significantly by 80.9%, 68.4%(P<0.001).
Conclusion In IRI group, levels of IMD in blood plasma and kidney increased significantly; and consistently there appeared marked increases of the mRNA expressions of IMD and of its receptors, and the protein expressions of IMD and CRLR in kidneys. These results suggest that IMD may have participated in the process of renal IRI, and may have important physiological and pathological significance in terms of kidney protection.
2. The influence of IMD exerted on the hypoxia/reoxygenation model of rat renal tubular epithelial cells and its mechanism
Objective The hypoxia/reoxygenation(H/R) injury models of rat renal tubular epithelial cells (NRK-52E), which were transfected the eukaryotic expression vector of rat IMD, were established to simulate IRI in vivo, to investigate the molecule mechanism of IMD cytoprotection.
Methods⑴To establish the H/R injury model of NRK-52E by regulating the pressure of N2 in incubator to hypoxia condition, the cells were cultured with hypoxia for 1h and then with reoxygenation for 1.5h. The models were evaluated by detecting living cell count, cell viability and the activity of lactate dehydrogenase (LDH) in the culture medium.⑵The full length IMD cDNA, which was synthesized artificially, was connected with pMD19-T Simple vector to form a cloned vector. This cloned vector was sub-cloned into pIRES2-EGFP to construct an eukaryotic expression vector, which is named pIRES2-EGFP/IMD. Restriction enzyme digestion and sequencing analysis were adopted to test the recombinant plasmids.⑶NRK-52E cells were transfected by transfection complex comprising optimal proportion of pIRES2-EGFP/IMD plasmid and Fugene HD reagents. Transfection efficiency was tested by observation for EGFP made by fluorescent microscope and flow cytometery (FCM) after 48h. Total RNA and protein was extracted from EGFP positive NRK-52E cells. IMD mRNA and protein expression level was evaluated by RT-PCR and Western-blotting. The cell groups with high transfection efficiency were screened for two weeks or so by incubating with medium containing G418(300μg/ml), and the positive cloning NRK-52E cells of stable expression IMD obtained. The expression of IMD protein in IMD plasmid group after screening was evaluated by Western-blotting.⑷The NRK-52E cells were divided into 7 groups. One of them was control group; the other six model groups were exposed to H/R condition, following the intervention of single H/R, primitive vector, highly expressed IMD vector, NADPH oxidase inhibitor(DPI), cyclic adenosine monophosphate(cAMP) analogue (8-Br-cAMP), PKA inhibitor(H-89), respectively. The contents of LDH and cAMP in the culture medium and the levels of NADPH oxidase, superoxide dismutase(SOD), malondialdehyde(MDA) and reactive oxygen species(ROS) in cells were detected to observe the influence of IMD on H/R injury and its molecule mechanism.
Results⑴After hypoxia 1h and reoxygenation 1.5h, cell count and cell viability decreased significantly and the activity of LDH increased significantly (P<0.05); the model was established successfully.⑵Restriction enzyme digestion and sequencing analysis showed that pMD19-T Simple/IMD cloning vector and pIRES2-EGFP/IMD eukaryotic expression vector had been constructed successfully.⑶NRK-52E cells was transfected with the optimal transfection proportion of pIRES2-EGFP/IMD to Fugene HD reagent as 3μg: 12μl, transfection efficiency evaluated by fluorescence microscope and FCM showed that the transfection efficiency increased with the prolongation of time and reached its peak 71.34±6.24% at 48h(P<0.01). The result of RT-PCR and Western blotting showed the expression levels of IMD mRNA and protein in IMD plasmid group were increased significantly (P<0.01 and P<0.001, respectively). The positive transfected cells after G418 screening were found clone-like proliferation on the third day, and the untransfected cells died in large bulk on the fifth day. Then the positive transfected cells increased gradually; and the positive cloning cells blending into slices were found on the fourteenth day, which occupied 60-70% of the total cell number. The result of Western blotting showed the expression of IMD protein in IMD plasmid group after screening increased significantly, which indicate the IMD expression of the cell is stable.⑷In NRK-52E cells following the seven treatments, the changes of detected index were shown:①Contrasted with the control group, the level of LDH of all model groups increased significantly (P<0.01); but the extent of increase in IMD plasmid group and 8-Br-cAMP group were smaller than that in H/R group(P﹤0.05).②Contrasted with the control group, the level of cAMP of all model groups also increased significantly(P<0.01), and IMD plasmid + H-89 group and 8-Br-cAMP group were exceptionally noticeable; when compared with H/R group, the two groups were the most remarkably increased groups(P﹤0.01), IMD plasmid group increased mildly and DPI group decreased(P﹤0.05).③Contrasted with the control group, the activity of NADPH oxidase of all model groups increased significantly except DPI group(P<0.01), but the extent of increase in 8-Br-cAMP group was the smallest. Compared with H/R group, the levels of NADPH oxidase of 8-Br-cAMP group and DPI group decreased by 33.92%(P﹤0.05) and 52.54%(P﹤0.01), respectively.④The content of MDA increased in H/R group (P﹤0.01), primitive plasmid group (P﹤0.01), IMD plasmid + H-89 group (P﹤0.05) and DPI group (P﹤0.01), but decreased in IMD plasmid group, IMD plasmid + H-89 group, 8-Br-cAMP group and DPI group(P﹤0.05) compared with H/R group.⑤The content of ROS increased in H/R group, primitive plasmid group, IMD plasmid + H-89 group (P﹤0.05), but decreased in IMD plasmid group compared with H/R group(P﹤0.05).⑥The content of SOD increased in IMD plasmid group, IMD plasmid + H-89 group and DPI group(P﹤0.05), but it changed little in the other four groups.
Conclusion In H/R model of NRK-52E, IMD could increase the level of cAMP via cAMP/PKA access, inhibit the activity of NADPH oxidase, decrease the production of ROS, upgrade SOD, alleviate the cell oxidative damage, regulate the oxidative and anti-oxidative balance. All these suggest that IMD, as an endogenous antioxidant, plays a protective role in cell protection during H/R-induced oxidative injury.
引文
[1] Shigehiko Uchino. The epidemiology of acute renal failure in the world[J]. Curr Opin Crit Care[J]. 2006;12(6):538-543.
[2] Hoste EA, Schurgers M. Epidemiology of acute kidney injury: how big is the problem[J]? Crit Care Med. 2008 Apr;36(4 Suppl):S146-S151.
[3] Lameire N, Van Biesen W, Vanholder R. Acute renal failure[J]. Lancet. 2005; 365(9457):417-430.
[4] Chatterjee PK. Novel pharmacological approaches to the treatment of renal ischemia-reperfusion injury: a comprehensive review[J]. Naunyn Schmiedebergs Arch Pharmacol. 2007;376(1-2):1-43.
[5] Jassem W, Heaton ND. The role of mitochondria in ischemia/reperfusion injury in organ transplantation[J]. Kidney Int. 2004;66(2):514-517.
[6] Schrier RW, Wang W, Poole B, et al. Acute renal failure: definitions, diagnosis, pathogenesis, and therapy[J]. J Clin Invest. 2004;114(1):5-14.
[7] Nishimatsu H, Hirata Y, Shindo T, et al. Role of endogenous adrenomedullin in the regulation of vascular tone and ischemic renal injury: studies on transgenic/knockout mice of adrenomedullin gene[J]. Circ Res. 2002;90(6): 657-663.
[8] Beltowski J, Jamroz A. Adrenomedullin-what do we know 10 years since its discovery[J]?Pol J Pharmacol. 2004;56(1):5-27.
[9] Roh J, Chang CL, Bhalla A, et al. Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complexes[J]. J Biol Chem. 2004; 279(8):7264-7274.
[10] Takei Y, Inoue K, Ogoshi M, et al. Identification of novel adrenomedullin in mammals: a potent cardiovascular and renal regulator[J]. FEBS Lett. 2004;556(1-3):53-58.
[11] Yang JH, Jia YX, Pan CS, et al. Effects of intermedin(1-53) on cardiac function and ischemia/reperfusion injury in isolated rat hearts[J]. Biochem Biophys Res Commun. 2005;327(3):713-719.
[12] McLatchie LM, Fraser NJ, Main MJ, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor[J]. Nature. 1998;393 (6683):333-339.
[13] Taylor MM, Bagley SL, Samson WK. Intermedin/adrenomedullin-2 acts within central nervous system to elevate blood pressure and inhibit food and water intake[J]. Am J Physiol Regul Integr Comp Physiol. 2005;288(4):R919-927.
[14] Takahashi K, Kikuchi K, Maruyama Y, et al. Immunocytochemical localization of adrenomedullin 2/intermedin-like immunoreactivity in human hypothalamus, heart and kidney[J]. Peptides. 2006;27(6):1383-1389.
[15] Morimoto R, Satoh F, Murakami O, et al. Expression of adrenomedullin2/ intermedin in human brain, heart, and kidney[J]. Peptides. 2007;28(5):1095- 1103.
[16] Fujisawa Y, Nagai Y, Miyatake A,et al. Renal effects of a new member of adrenomedullin family, adrenomedullin2, in rats[J]. Eur J Pharmacol. 2004; 497(1):75-80.
[17] Yang JH, Qi YF, Jia YX, et al. Protective effects of intermedin/adrenomedullin2 on ischemia/reperfusion injury in isolated rat hearts[J]. Peptides. 2005;26(3): 501-507.
[18] Jia YX, Yang JH, Pan CS, et al. Intermedin1-53 protects the heart against isoproterenol-induced ischemic injury in rats[J]. Eur J Pharmacol. 2006;549(1-3): 117-123.
[19] Bell D, Zhao Y, McCoy FGP, et al. Differential effects of an anti-oxidant intervention on cardiomyocyte expression of adrenomedullin and intermedin and their receptor components in chronic nitric oxide deficiency[J]. Cell Physiol Biochem.2007;20(5): 269-282.
[20] Hirose T, Totsune K, Mori N, et al. Increased expression of adrenomedullin 2/intermedin in rat hearts with congestive heart failure[J]. Eur J Heart Fail. 2008;10(9):840-849.
[21] Zeng Q, Yuan Y, Wang X, et al. Upregulated expression of intermedin and its receptor in the myocardium and aorta in spontaneously hypertensive rats[J]. Peptides. 2009;30(2):391-399.
[22] Bonventre JV, Weinberg JM. Weinberg. Recent advances in the pathophysiology of ischemic acute renal failure[J]. J Am Soc Nephrol. 2003;14(8): 2199-2210.
[23] Devarajan P. Cellular and molecular derangements in acute tubular necrosis[J]. Curr Opin Pediatr. 2005;7(2):193-199.
[24] Chien CT, Lee PH, Chen CF,et al. De novo demonstration and co-localization of free-radical production and apoptosis formation in rat kidney subjected to ischemia/reperfusion[J]. J Am Soc Nephrol. 2001;12(5):973-982.
[25] Donnahoo KK, Shames BD, Harken AH, et al. Review article: the role of tumor necrosis factor in renal ischemia-reperfusion injury[J]. J Urol. 1999;162(1): 196-203.
[26] Yoshimoto T, Fukai N, Sato R, et al. Antioxidant effect of adrenomedullin on angiotensin II-induced reactive oxygen species generation in vascular smooth muscle cells[J]. Endocrinology. 2004;145(7):3331-3337.
[27] Chen L, Kis B, Hashimoto H, et al. Adrenomedullin 2 protects rat cerebral endothelial cells from oxidative damage in vitro[J]. Brain Res. 2006;1086(1): 42-49.
[1] Melnikov VY, Faubel S, Siegmund B, et al. Neutrophil-independent mechanisms of caspase-1- and IL-18-mediated ischemic acute tubular necrosis in mice[J]. J Clin Invest. 2002;110(8):1083-1091.
[2] Thadhani R, Dascual M, Bonventre JV. Acute renal failure[J]. N Eng1 J Med, 1996,334(22):1448-1460.
[3] Roh J, Chang CL, Bhalla A, et al. Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity- modifying protein receptor complexes[J]. J Biol Chem. 2004;279(8):7264-7274.
[4] Takei Y, Inoue K, Ogoshi M, et al. Identification of novel adrenomedullin in mammals: a potent cardiovascular and renal regulator[J]. FEBS Lett. 2004;556(1-3):53-58.
[5] Beltowski J, Jamroz A. Adrenomedullin-what do we know 10 years since its discovery[J]?Pol J Pharmacol. 2004;56(1):5-27.
[6] Nishimatsu H, Hirata Y, Shindo T, et al. Role of endogenous adrenomedullin in the regulation of vascular tone and ischemic renal injury: studies on transgenic/knockout mice of adrenomedullin gene[J]. Circ Res. 2002;90(6):657-663.
[7] Takahashi K, Kikuchi K, Maruyama Y, et al. Immunocytochemical localization of adrenomedullin 2/intermedin-like immunoreactivity in human hypothalamus, heart and kidney[J]. Peptides. 2006;27(6):1383-1389.
[8] Morimoto R, Satoh F, Murakami O, et al. Expression of adrenomedullin2/intermedin in human brain, heart, and kidney[J]. Peptides. 2007;28(5):1095-1103.
[9] Fujisawa Y, Nagai Y, Miyatake A,et al. Renal effects of a new member of adrenomedullin family, adrenomedullin2, in rats[J]. Eur J Pharmacol. 2004;497(1):75-80.
[10] Dong YL, Vegiraju S, Chauhan M, et al. Expression of calcitonin gene-related peptide receptor components, calcitonin receptor-like receptor and receptor activity modifying protein 1, in the rat placenta during pregnancy and their cellular localization[J]. Mol Hum Reprod. 2003, 9(8):481-490.
[11] McLatchie LM, Fraser NJ, Main MJ, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor–like receptor[J]. Nature. 1998;393(6683): 333-339.
[12]陈斌,石洪波,张雪军,等. Intermedin预处理对大鼠肾缺血离体再灌注损伤的保护作用[J].临床泌尿外科杂志. 2008;23(5):385-388.
[13] Hagiwara M, Bledsoe G, Yang ZR, et al. Intermedin ameliorates vascular and renal injury by inhibition of oxidative stress[J]. Am J Physiol Renal Physiol. 2008;295(6):F1735- F1743.
[14] Bell D, McDermott BJ. Intermedin (adrenomedullin-2): a novel counter-regulatory peptide in the cardiovascular and renal systems[J]. Br J Pharmacol. 2008;153(Suppl 1):S247-S262.
[15] Zeng Q, Yuan Y, Wang X, et al. Upregulated expression of intermedin and its receptor in the myocardium and aorta in spontaneously hypertensive rats[J]. Peptides. 2009;30(2):391-399.
[16] Sandner P, Hofbauer KH, Expression of adrenomedullin in hypoxic and ischemic rat kidneys and human kidneys with arterial stenosis[J]. Am J Physiol Regul Integr Comp Physiol. 2004;286(5):R942-R951.
[17] Carrizo GJ, Wu R, Cui X, et al. Adrenomedullin and adrenomedullin-binding protein-1 downregulate inflammatory cytokines and attenuate tissue injury after gut ischemia- reperfusion[J]. Surgery. 2007;141(2):245-253.
[18]贾月霞,李桂忠,杨靖辉,等.急性心力衰竭大鼠心肌intermedin受体的变化[J].宁夏医学院学报. 2005;27(5):337-339.
[19] Jia YX, Yang JH, Pan CS, et al. Intermedin1-53 protects the heart against isoproterenol-induced ischemic injury in rats[J]. Eur J Pharmacol. 2006;549(1-3):117-123.
[20] Zhao Y, Bell D, Smith LR, et al. Differential effects of components of the cardiomyocyte adrenomedullin/intermedin receptor system following blood pressure reduction in nitric oxide-deficient hypertension[J]. J Pharmacol Exp Ther. 2006;316(3): 1269-1281.
[21] Bell D, Zhao Y, Devine AB, et al. Influence of atenolol and nifedipine on nitric-oxide deficient cardiomyocyte hypertrophy and expression of the cardio-endocrine peptide intermedin and its receptor components[J]. Cell Physiol Biochem. 2008;21(1-3):203-214.
[22] Dong F, Taylor MM, Samson WK, et al. Intermedin (adrenomedullin-2) enhances cardiac contractile function via a protein kinase C- and protein kinase A- dependent pathway in murine ventricular myocytes [J]. J Appl Physiol. 2006;101(3):778-784.
[23] Yang JH, Qi YF, Jia YX, et al. Protective effects of intermedin/adrenomedullin2 on ischemia/reperfusion injury in isolated rat hearts[J]. Peptides. 2005;26(3):501-507.
[24] Yang JH, Pan CS, Jia YX, et al. Intermedin 1-53 activates L-arginine/nitric oxide synthase/nitric oxide pathway in rat aortas[J]. Biochem Biophys Res Commun. 2006; 341(2):567-572.
[25] Chauhan M, Ross GR, Yallampalli U, et al. Adrenomedullin-2, a novel calcitonin/calcitonin-gene-related peptide family peptide, relaxes rat mesenteric artery: influence of pregnancy[J]. Endocrinology. 2007;148(4):1727-1735.
[26] Chen L, Kis B, Hashimoto H, et al. Adrenomedullin 2 protects rat cerebral endothelial cells from oxidative damage in vitro[J]. Brain Res. 2006;1086(1):42-49.
[27] Hagiwara M, Bledsoe G, Yang ZR, et al. Intermedin ameliorates vascular and renal injury by inhibition of oxidative stress[J]. Am J Physiol Renal Physiol. 2008;295(6):F1735- F1743.
[1] Koyama T, Temma K, Akera T. Reperfusion-induced contracture develops with a decreasing [Ca2+]i in single heart cells[J]. Am J Physiol. 1991;261(4 Pt 2):H1115-H1122.
[2] Basnakian AG, Ueda N, Hong X, et al. Ceramide synthase is essential for endonuclease-mediated death of renal tubular epithelial cells induced by hypoxia- reoxygenation[J]. Am J Physiol Renal Physiol. 2005;288(2):F308-F314.
[3] Combe C, Burton CJ, Dufourco P, et al. Hypoxia induces intercellular adhesion molecule-1 on cultured human tubular cells[J]. Kidney Int. 1997;51(6): 1703-1709.
[4] Doctor RB, Mandel LJ. Minimal role of xanthine oxidase and oxygen free radicals in rat renal tubular reoxygenation injury[J].J Am Soc Nephrol. 1991;1(7):959-969.
[5] Sandy Primrose, Richard Twyman, Bob Old. Principles of Gene Manipulation, Sixth Edition[M].影印版.高等教育出版社. 2002:43-85.
[6] Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP)[J]. Gene. 1996;173(1 Spec No):33-38.
[7]曹亚.实用分子生物学操作指南[M].北京:人民卫生出版社. 2003:185.
[8] Hu QH, Liang WQ. A comparative study of electroporation and iontophoresis for percutaneous penetration of naproxen[J]. Pharmazie. 2003;58(3):192-194.
[9] Jaroszeski MJ, Heller LC, Gilbert R, et al. Electrically mediated plasmid DNA delivery to solid tumors in vivo[J]. Methods Mol Biol. 2004;245:237-244.
[10] Chrenek P, Vasicek D, Makarevich AV, et al. Increased transgene integration efficiency upon microinjection of DNA into both pronuclei of rabbit embryos[J]. Transgenic Res. 2005;14(4):417-428.
[11] Koike H, Tomita N, Azuma H, et al. An efficient gene transfer method mediated by ultrasound and microbubbles into the kidney[J]. J Gene Med. 2005;7(1): 108-116.
[12] Guo DP, Li XY, Sun P, et al. Ultrasound/microbubble enhances foreign gene expression in ECV304 cells and murine myocardium[J]. Acta Biochim Biophys Sin (Shanghai). 2004;36(12):824-831.
[13] Sambrook J, Fritsch EF, Maniaatis T, et al. Molecular cloning, A laboratory mannual[M]. 2nd ed, NewYork: Cold Spring Harbor. 1995:345-347.
[14] Suqiyama M, Matsuura M, Takeuchi Y, et al. Possible mechanism of polycation liposome(PCL)-mediated gene transfer[J]. Biochim Biophys Acta. 2004;1660(1-2): 24-30.
[15] Delenda C. Lentiviral vectors: optimization of packaging, transduction and gene expression[J]. J Gene Med. 2004;6(Suppl 1):S125-S138.
[16] Ghosh SS, Gopinath P, Ramesh A. Adenoviral vectors: a promising tool for gene therapy[J]. Appl Biochem Biotechnol. 2006;133(1):9-29.
[17] Schrier RW, Wang W, Poole B, et al. Acute renal failure: definitions, diagnosis, pathogenesis, and therapy[J]. J Clin Invest. 2004; 114(1):5-14.
[18] Bonventre JV, Weinberg JM. Weinberg. Recent advances in the pathophysiology of ischemic acute renal failure[J]. J Am Soc Nephrol. 2003;14(8): 2199-2210.
[19] Chien CT, Lee PH, Chen CF,et al. De novo demonstration and co-localization of free-radical production and apoptosis formation in rat kidney subjected to ischemia/reperfusion[J]. J Am Soc Nephrol. 2001;12(5):973-982.
[20] Donnahoo KK, Shames BD, Harken AH, et al. Review article: the role of tumor necrosis factor in renal ischemia-reperfusion injury[J]. J Urol. 1999;162(1): 196-203.
[21] Yang JH, Qi YF, Jia YX, et al. Protective effects of intermedin/adrenomedullin2 on ischemia/reperfusion injury in isolated rat hearts[J]. Peptides. 2005; 26(3): 501-507.
[22] Yang JH, Jia YX, Pan CS, et al. Effects of intermedin(1-53) on cardiac function and ischemia/reperfusion injury in isolated rat hearts[J]. Biochem Biophys Res Commun. 2005;327(3):713-719.
[23] Yoshimoto T, Fukai N, Sato R, et al. Antioxidant effect of adrenomedullin on angiotensin II-induced reactive oxygen species generation in vascular smooth muscle cells[J]. Endocrinology. 2004;145(7):3331-3337.
[24] Versteilen AM, Di Maggio F, Leemreis JR, et al. Molecular mechanisms of acute renal failure following ischemia/reperfusion[J]. Int J Artif Organs. 2004;27(12): 1019-1029.
[25] Wang Q, Tang XN, Yenari MA. The inflammatory response in stroke[J]. J Neuroimmunol. 2007;184(1-2):53-68.
[26] Sharma S, Dewald O, Adrogue J, et al. Induction of antioxidant gene expression in a mouse model of ischemic cardiomyopathy is dependent on reactive oxygen species[J]. Free Radic Biol Med. 2006;40(12):2223-2231.
[27] Thurman JM. Triggers of inflammation after renal ischemia/reperfusion[J]. Clin Immunol. 2007;123(1):7-13.
[28] Halliwell B, Gutteridge JM, Cross CE. Free radicals, antioxidants, and human disease: where are we now?[J]. J Lab Clin Med. 1992;119(6):598-620.
[29]陈瑗,周玫.自由基医学基础与病理生理[M].北京:人民卫生出版社. 2002:3-52.
[30] Chen L, Kis B, Hashimoto H, et al. Adrenomedullin 2 protects rat cerebral endothelial cells from oxidative damage in vitro[J]. Brain Res. 2006;1086(1): 42-49.
[31] Nogueira-Machado JA, Lima e Silva FC, Medina LO, et al. Modulation of the reactiveoxygen species (ROS) generation mediated by cyclic AMP-elevating agents or Interleukin 10 in granulocytes from type 2 diabetic patients (NIDDM): a PKA-independent phenomenon[J]. Diabetes Metab. 2003;29(5):533-537.
[1] Wimalawansa SJ. Amylin, calcitonin gene-related peptide, calcitonin, and adrenomedullin: a peptide superfamily[J]. Crit Rev Neurobiol. 1997;11(2-3): 167-239.
[2] Roh J, Chang CL, Bhalla A, et al. Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complexes[J]. J Biol Chem. 2004;279(8):7264-7274.
[3] Takei Y, Inoue K, Ogoshi M, et al. Identification of novel adrenomedullin in mammals: a potent cardiovascular and renal regulator[J]. FEBS Lett. 2004;556 (1-3):53-58.
[4] Yang JH, Jia YX, Pan CS, et al. Effects of intermedin(1-53) on cardiac function and ischemia/reperfusion injury in isolated rat hearts[J]. Biochem Biophys Res Commun. 2005;327(3):713-719.
[5] Chang CL, Roh J, Hsu SY. Intermedin, a novel calcitonin family peptide that exists in teleosts as well as in mammals: a comparison with other calcitonin/ intermedin family peptides in vertebrates[J]. Peptides. 2004;25(10): 1633-1642.
[6] Poyner DR, Sexton PM, Marshall I, et al. International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors[J]. Pharmacol Rev. 2002;54(2):233-246.
[7] Kindt F, Wiegand S, Loser C, et al. Intermedin: a skin peptide that is downregulated in atopic dermatitis[J]. J Invest Dermatol. 2007;127(3):605-613.
[8] Takahashi K, Kikuchi K, Maruyama Y, et al. Immunocytochemical localization of adrenomedullin 2/intermedin-like immunoreactivity in human hypothalamus, heart and kidney[J]. Peptides. 2006;27(6):1383-1389.
[9] Takei Y, Hyodo S, Katafuchi T, et al. Novel fish-derived adrenomedullin in mammals: structure and possible function[J]. Peptides. 2004;25(10):1643-1656.
[10] Pan CS, Yang JH, Cai DY, et al. Cardiovascular effects of newly discovered peptide intermedin/adrenomedullin-2[J]. Peptides. 2005;26(9):1640-1646.
[11] Zhao Y, Bell D, Smith LR, et al. Differential effects of components of the cardiomyocyte adrenomedullin/intermedin receptor system following blood pressure reduction in nitric oxide-deficient hypertension[J]. J Pharmacol Exp Ther. 2006;316(3): 1269-1281.
[12] Morimoto R, Satoh F, Murakami O, et al. Expression of adrenomedullin2/ intermedin in human brain, heart, and kidney[J]. Peptides. 2007;28(5):1095-1103.
[13] McLatchie LM, Fraser NJ, Main MJ, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor–like receptor[J]. Nature. 1998;393 (6683): 333-339.
[14] Yang JH, Qi YF, Jia YX, et al. Protective effect s of intermedin/adrenomedullin2 on ischemia/ reperfusion injury in isolated rat hearts[J]. Peptides. 2005;26(3): 501-507.
[15] Jia YX, Yang JH, Pan CS, et al. Intermedin1-53 protects the heart against isoproterenol-induced ischemic injury in rats[J]. Eur J Pharmacol. 2006;549(1-3): 117-123.
[16] Dong F, Taylor MM, Samson WK, et al. Intermedin (adrenomedullin-2) enhances cardiac contractile function via a protein kinase C- and protein kinase A- dependent pathway in murine ventricular myocytes [J]. J Appl Physiol. 2006;101(3):778-784.
[17] Beltowski J, Jamroz A. Adrenomedullin-what do we know 10 years since its discovery[J]?Pol J Pharmacol. 2004;56(1):5-27.
[18] Terata K, Miura H, Liu Y, et al. Human coronary arteriolar dilation to adrenomedullin: role of nitric oxide and K(+) channels[J]. Am J Physiol Heart Circ Physiol. 2000;279(6): H2620-H2626.
[19] Sata M, Kakoki M, Nagata D, et al. Adrenomedullin and nitric oxide inhibit human endothelial cell apoptosis via acyclic GMP2 independent mechanism[J]. Hypertension. 2000;36(1):83-88.
[20] Yang JH, Pan CS, Jia YX, et al. Intermedin 1-53 activates L-arginine/nitric oxide synthase/nitric oxide pathway in rat aortas[J]. Biochem Biophys Res Commun. 2006;341(2):567-572.
[21] Chauhan M, Ross GR, Yallampalli U, et al. Adrenomedullin-2, a novel calcitonin/calcitonin-gene-related peptide family peptide, relaxes rat mesenteric artery: influence of pregnancy[J]. Endocrinology. 2007;148(4):1727-1735.
[22] Kandilci HB, Gumusel B, Lippton H. Intermedin/adrenomedullin-2 (IMD/AM2) relaxes rat main pulmonary arterial rings via cGMP-dependent pathway: role of nitric oxide and large conductance calcium-activated potassium channels (BK(Ca))[J]. Peptides. 2008;29 (8):1321-1328.
[23] Taylor MM, Bagley SL, Samson WK. Intermedin/adrenomedullin-2 acts within central nervous system to elevate blood pressure and inhibit food and water intake[J]. Am J Physiol Regul Integr Comp Physiol. 2005;288(4):R919-R927.
[24] Ren YS, Yang JH, Zhang J, et al. Intermedin 1-53 in central nervous system elevates arterial blood pressure in rats[J]. Peptides. 2006;27(1):74-79.
[25] Cui H, Kohsaka A, Waki H, et al. Adrenomedullin 2 microinjection into the nucleus tractus solitarius elevates arterial pressure and heart rate in rats[J]. Auton Neurosci. 2008;142(1-2):45-50.
[26] Charles CJ, Rademaker MT, Richards AM. Hemodynamic, hormonal, and renal actions ofadrenomedullin-2 in normal conscious sheep[J]. Endocrinology. 2006; 147(4):1871-1877.
[27] Abdelrahman AM, Pang CC. Effect of intermedin/adrenomedullin-2 on venous tone in conscious rats[J]. Naunyn Schmiedebergs Arch Pharmacol. 2006;373(5): 376-380.
[28] Fujisawa Y, Nagai Y, Miyatake A, et al. Renal effects of a new member of adrenomedullin family, adrenomedullin2, in rats[J]. Eur J Pharmacol. 2004;497(1): 75-80.
[29] Fujisawa Y, Nagai Y, Miyatake A, et al. Roles of adrenomedullin2 in regulating the cardiovascular and sympathetic nervous systems in conscious rats[J]. Am J Physiol Heart Circ Physiol. 2006;59(3):H1120-H1127.
[30] Fujisawa Y, Nagai Y, Miyatake A, et al. Effects of adrenomedullin2 on regional hemodynamics in conscious rats[J]. Eur J Pharmacol. 2007;558(1-3):128-132.
[31]王曦,曾强,袁鹰,等.肾上腺髓质素2对自发性高血压大鼠血压和心功能的影响[J].中华高血压杂志. 2007;15(12):1012-1016.
[32] Kobayashi Y, Liu YJ, Gonda T, et al. Coronary vasodilatory response to a novel peptide, adrenomedullin 2[J]. Clin Exp Pharmacol Physiol. 2004:31(Suppl 2):S49-S50.
[33] Doganci S, Yildirim V, Bolcal C, et al. Intermedin (IMD/AM2) dilates the pig coronary vascular bed through release of nitric oxide[J]. FASEB J. 2007:21(6): 1380-1385.
[34] Bell D, Zhao Y, McCoy FGP, et al. Differential effects of an anti-oxidant intervention on cardiomyocyte expression of adrenomedullin and intermedin and their receptor components in chronic nitric oxide deficiency[J]. Cell Physiol Biochem.2007;20(5): 269-282.
[35] Bell D, Zhao Y, Devine AB, et al. Influence of atenolol and nifedipine on nitric-oxide deficient cardiomyocyte hypertrophy and expression of the cardio-endocrine peptide intermedin and its receptor components[J]. Cell Physiol Biochem. 2008;21(1-3):203-214.
[36] Bell D, Zhao Y, McCoy FP, et al. Expression of the counter-regulatory peptide intermedin is augmented in the presence of oxidative stress in hypertrophied cardiomyocytes[J]. Cell Physiol Biochem. 2008;21(5-6):409-420.
[37] Zeng Q, Yuan Y, Wang X, et al. Upregulated expression of intermedin and its receptor in the myocardium and aorta in spontaneously hypertensive rats[J]. Peptides. 2009;30(2): 391-399.
[38] Hirose T, Totsune K, Mori N, et al. Increased expression of adrenomedullin 2/intermedin in rat hearts with congestive heart failure[J]. Eur J Heart Fail. 2008;10(9):840-849.
[39]张玉青,黄卡特,毛孙忠,等.高脂血症和高脂血症性脂肪肝大鼠肾上腺髓质素2的变化[J].世界华人消化杂志. 2007;15(21):2341-2344.
[40]陈斌,石洪波,张雪军,等. Intermedin预处理对大鼠肾缺血离体再灌注损伤的保护作用[J].临床泌尿外科杂志. 2008;23(5):385-388.
[41] Hagiwara M, Bledsoe G, Yang ZR, et al. Intermedin ameliorates vascular and renal injury by inhibition of oxidative stress[J]. Am J Physiol Renal Physiol. 2008;295(6): F1735-F1743.
[42] Burak Kandilci H, Gumusel B, Wasserman A, et al. Intermedin/adrenomedullin-2 dilates the rat pulmonary vascular bed: dependence on CGRP receptors and nitric oxide release[J]. Peptides. 2006;27(6):1390-1396.
[43]于晓敏,刘新民,齐永芬,等.中介素1-53对油酸致大鼠急性肺损伤的保护作用[J].中华结核和呼吸杂志. 2006;29(12):808-811.
[44]于晓敏,刘新民,齐永芬,等.中介素及其受体系统在油酸致大鼠急性肺损伤中的变化和意义[J].北京大学学报(医学版). 2006;38(5):496-500.
[45] Gong YS, Fan XF, Wu XM, et al. Changes of intermedin/adrenomedullin 2 and its receptors in the right ventricle of rats with chronic hypoxic pulmonary hypertension[J]. Sheng Li Xue Bao. 2007;59(2):210-214.
[46]刘青,王新均,董有平,等.败血症休克大鼠肺组织Intermedin含量及其mRNA表达[J].郧阳医学院学报. 2007;26(2):74-77.
[47]吴东红,田丽鲜,范小芳,等.低氧对大鼠肺组织中介素及其受体系统表达的影响[J].中国微循环. 2007;11(2):94-97.
[48]范小芳,黄萍,龚永生,等.慢性低氧性肺动脉高压大鼠肺组织肾上腺髓质素2的变化[J].中国应用生理学杂志. 2007;23(4):467-471.
[49] Hashimoto H, Hyodo S, Kawasaki M, et al. Centrally administered adrenomedullin 2 activates hypothalamic oxytocin-secreting neurons, causing elevated plasma oxytocin level in rats[J]. Am J Physiol Endocrinol Metab. 2005;289(5):E753-E761.
[50] Hashimoto H, Hyodo S, Kawasaki M, et al. Adrenomedullin 2 (AM2)/intermedin is a more potent activator of hypothalamic oxytocin-secreting neurons than AM possibly through an unidentified receptor in rats. Peptides[J]. 2007;28(5): 1104-1012.
[51] Taylor MM, Samson WK. Stress hormone secretion is altered by central administration of intermedin/adrenomedullin-2[J]. Brain Res. 2005;1045(1-2): 199-205.
[52] Taylor MM, Bagley SL, Samson WK. Intermedin/Adrenomedullin-2 inhibits growth hormone release from cultured, primary anterior pituitary cells[J]. Endocrinology. 2006;147(2):859-864.
[53] Lin Chang C, Roh J, Park JI, et al. Intermedin functions as a pituitary paracrine factor regulating prolactin release[J]. Mol Endocrinol. 2005;19(11):2824-2838.
[54] Chen L, Kis B, Hashimoto H, et al. Adrenomedullin 2 protects rat cerebral endothelial cellsfrom oxidative damage in vitro[J]. Brain Res. 2006;1086(1): 42-49.
[55]蔡莉蓉,李玉珍,刘秀华.肾上腺髓质素2对大鼠脑微血管内皮细胞增殖的影响[J].中国病理生理杂志. 2006;22(5):908-910.
[56] White MM, Samson WK. Intermedin 17-47 does not function as a full intermedin antagonist within the central nervous system or pituitary[J]. Peptides. 2007;28(11): 2171-2178.
[57] Owji AA, Chabot JG, Dumont Y, et al. Adrenomedullin-2/intermedin induces cAMP accumulation in dissociated rat spinal cord cells: evidence for the existence of a distinct class of sites of action[J]. J Mol Neurosci. 2008;35(3):355-361.
[58] Nishikimi T. Adrenomedullin in the kidney-renal physiological and pathophysiological roles[J]. Curr Med Chem. 2007;14(15):1689-1699.
[59] Nishimatsu H, Hirata Y, Shindo T, et al. Role of endogenous adrenomedullin in the regulation of vascular tone and ischemic renal injury: studies on transgenic/knockout mice of adrenomedullin gene[J]. Circ Res. 2002;90(6): 657-663.
[60] Chao J, Kato K, Zhang JJ, et al. Human adrenomedullin gene delivery protects against cardiovascular remodeling and renal injury[J]. Peptides. 2001;22(11): 1731-1737.
[2] Hoste EA, Schurgers M. Epidemiology of acute kidney injury: how big is the problem[J]? Crit Care Med. 2008 Apr;36(4 Suppl):S146-S151.
[3] Lameire N, Van Biesen W, Vanholder R. Acute renal failure[J]. Lancet. 2005; 365(9457):417-430.
[4] Chatterjee PK. Novel pharmacological approaches to the treatment of renal ischemia-reperfusion injury: a comprehensive review[J]. Naunyn Schmiedebergs Arch Pharmacol. 2007;376(1-2):1-43.
[5] Jassem W, Heaton ND. The role of mitochondria in ischemia/reperfusion injury in organ transplantation[J]. Kidney Int. 2004;66(2):514-517.
[6] Schrier RW, Wang W, Poole B, et al. Acute renal failure: definitions, diagnosis, pathogenesis, and therapy[J]. J Clin Invest. 2004;114(1):5-14.
[7] Nishimatsu H, Hirata Y, Shindo T, et al. Role of endogenous adrenomedullin in the regulation of vascular tone and ischemic renal injury: studies on transgenic/knockout mice of adrenomedullin gene[J]. Circ Res. 2002;90(6): 657-663.
[8] Beltowski J, Jamroz A. Adrenomedullin-what do we know 10 years since its discovery[J]?Pol J Pharmacol. 2004;56(1):5-27.
[9] Roh J, Chang CL, Bhalla A, et al. Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complexes[J]. J Biol Chem. 2004; 279(8):7264-7274.
[10] Takei Y, Inoue K, Ogoshi M, et al. Identification of novel adrenomedullin in mammals: a potent cardiovascular and renal regulator[J]. FEBS Lett. 2004;556(1-3):53-58.
[11] Yang JH, Jia YX, Pan CS, et al. Effects of intermedin(1-53) on cardiac function and ischemia/reperfusion injury in isolated rat hearts[J]. Biochem Biophys Res Commun. 2005;327(3):713-719.
[12] McLatchie LM, Fraser NJ, Main MJ, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor[J]. Nature. 1998;393 (6683):333-339.
[13] Taylor MM, Bagley SL, Samson WK. Intermedin/adrenomedullin-2 acts within central nervous system to elevate blood pressure and inhibit food and water intake[J]. Am J Physiol Regul Integr Comp Physiol. 2005;288(4):R919-927.
[14] Takahashi K, Kikuchi K, Maruyama Y, et al. Immunocytochemical localization of adrenomedullin 2/intermedin-like immunoreactivity in human hypothalamus, heart and kidney[J]. Peptides. 2006;27(6):1383-1389.
[15] Morimoto R, Satoh F, Murakami O, et al. Expression of adrenomedullin2/ intermedin in human brain, heart, and kidney[J]. Peptides. 2007;28(5):1095- 1103.
[16] Fujisawa Y, Nagai Y, Miyatake A,et al. Renal effects of a new member of adrenomedullin family, adrenomedullin2, in rats[J]. Eur J Pharmacol. 2004; 497(1):75-80.
[17] Yang JH, Qi YF, Jia YX, et al. Protective effects of intermedin/adrenomedullin2 on ischemia/reperfusion injury in isolated rat hearts[J]. Peptides. 2005;26(3): 501-507.
[18] Jia YX, Yang JH, Pan CS, et al. Intermedin1-53 protects the heart against isoproterenol-induced ischemic injury in rats[J]. Eur J Pharmacol. 2006;549(1-3): 117-123.
[19] Bell D, Zhao Y, McCoy FGP, et al. Differential effects of an anti-oxidant intervention on cardiomyocyte expression of adrenomedullin and intermedin and their receptor components in chronic nitric oxide deficiency[J]. Cell Physiol Biochem.2007;20(5): 269-282.
[20] Hirose T, Totsune K, Mori N, et al. Increased expression of adrenomedullin 2/intermedin in rat hearts with congestive heart failure[J]. Eur J Heart Fail. 2008;10(9):840-849.
[21] Zeng Q, Yuan Y, Wang X, et al. Upregulated expression of intermedin and its receptor in the myocardium and aorta in spontaneously hypertensive rats[J]. Peptides. 2009;30(2):391-399.
[22] Bonventre JV, Weinberg JM. Weinberg. Recent advances in the pathophysiology of ischemic acute renal failure[J]. J Am Soc Nephrol. 2003;14(8): 2199-2210.
[23] Devarajan P. Cellular and molecular derangements in acute tubular necrosis[J]. Curr Opin Pediatr. 2005;7(2):193-199.
[24] Chien CT, Lee PH, Chen CF,et al. De novo demonstration and co-localization of free-radical production and apoptosis formation in rat kidney subjected to ischemia/reperfusion[J]. J Am Soc Nephrol. 2001;12(5):973-982.
[25] Donnahoo KK, Shames BD, Harken AH, et al. Review article: the role of tumor necrosis factor in renal ischemia-reperfusion injury[J]. J Urol. 1999;162(1): 196-203.
[26] Yoshimoto T, Fukai N, Sato R, et al. Antioxidant effect of adrenomedullin on angiotensin II-induced reactive oxygen species generation in vascular smooth muscle cells[J]. Endocrinology. 2004;145(7):3331-3337.
[27] Chen L, Kis B, Hashimoto H, et al. Adrenomedullin 2 protects rat cerebral endothelial cells from oxidative damage in vitro[J]. Brain Res. 2006;1086(1): 42-49.
[1] Melnikov VY, Faubel S, Siegmund B, et al. Neutrophil-independent mechanisms of caspase-1- and IL-18-mediated ischemic acute tubular necrosis in mice[J]. J Clin Invest. 2002;110(8):1083-1091.
[2] Thadhani R, Dascual M, Bonventre JV. Acute renal failure[J]. N Eng1 J Med, 1996,334(22):1448-1460.
[3] Roh J, Chang CL, Bhalla A, et al. Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity- modifying protein receptor complexes[J]. J Biol Chem. 2004;279(8):7264-7274.
[4] Takei Y, Inoue K, Ogoshi M, et al. Identification of novel adrenomedullin in mammals: a potent cardiovascular and renal regulator[J]. FEBS Lett. 2004;556(1-3):53-58.
[5] Beltowski J, Jamroz A. Adrenomedullin-what do we know 10 years since its discovery[J]?Pol J Pharmacol. 2004;56(1):5-27.
[6] Nishimatsu H, Hirata Y, Shindo T, et al. Role of endogenous adrenomedullin in the regulation of vascular tone and ischemic renal injury: studies on transgenic/knockout mice of adrenomedullin gene[J]. Circ Res. 2002;90(6):657-663.
[7] Takahashi K, Kikuchi K, Maruyama Y, et al. Immunocytochemical localization of adrenomedullin 2/intermedin-like immunoreactivity in human hypothalamus, heart and kidney[J]. Peptides. 2006;27(6):1383-1389.
[8] Morimoto R, Satoh F, Murakami O, et al. Expression of adrenomedullin2/intermedin in human brain, heart, and kidney[J]. Peptides. 2007;28(5):1095-1103.
[9] Fujisawa Y, Nagai Y, Miyatake A,et al. Renal effects of a new member of adrenomedullin family, adrenomedullin2, in rats[J]. Eur J Pharmacol. 2004;497(1):75-80.
[10] Dong YL, Vegiraju S, Chauhan M, et al. Expression of calcitonin gene-related peptide receptor components, calcitonin receptor-like receptor and receptor activity modifying protein 1, in the rat placenta during pregnancy and their cellular localization[J]. Mol Hum Reprod. 2003, 9(8):481-490.
[11] McLatchie LM, Fraser NJ, Main MJ, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor–like receptor[J]. Nature. 1998;393(6683): 333-339.
[12]陈斌,石洪波,张雪军,等. Intermedin预处理对大鼠肾缺血离体再灌注损伤的保护作用[J].临床泌尿外科杂志. 2008;23(5):385-388.
[13] Hagiwara M, Bledsoe G, Yang ZR, et al. Intermedin ameliorates vascular and renal injury by inhibition of oxidative stress[J]. Am J Physiol Renal Physiol. 2008;295(6):F1735- F1743.
[14] Bell D, McDermott BJ. Intermedin (adrenomedullin-2): a novel counter-regulatory peptide in the cardiovascular and renal systems[J]. Br J Pharmacol. 2008;153(Suppl 1):S247-S262.
[15] Zeng Q, Yuan Y, Wang X, et al. Upregulated expression of intermedin and its receptor in the myocardium and aorta in spontaneously hypertensive rats[J]. Peptides. 2009;30(2):391-399.
[16] Sandner P, Hofbauer KH, Expression of adrenomedullin in hypoxic and ischemic rat kidneys and human kidneys with arterial stenosis[J]. Am J Physiol Regul Integr Comp Physiol. 2004;286(5):R942-R951.
[17] Carrizo GJ, Wu R, Cui X, et al. Adrenomedullin and adrenomedullin-binding protein-1 downregulate inflammatory cytokines and attenuate tissue injury after gut ischemia- reperfusion[J]. Surgery. 2007;141(2):245-253.
[18]贾月霞,李桂忠,杨靖辉,等.急性心力衰竭大鼠心肌intermedin受体的变化[J].宁夏医学院学报. 2005;27(5):337-339.
[19] Jia YX, Yang JH, Pan CS, et al. Intermedin1-53 protects the heart against isoproterenol-induced ischemic injury in rats[J]. Eur J Pharmacol. 2006;549(1-3):117-123.
[20] Zhao Y, Bell D, Smith LR, et al. Differential effects of components of the cardiomyocyte adrenomedullin/intermedin receptor system following blood pressure reduction in nitric oxide-deficient hypertension[J]. J Pharmacol Exp Ther. 2006;316(3): 1269-1281.
[21] Bell D, Zhao Y, Devine AB, et al. Influence of atenolol and nifedipine on nitric-oxide deficient cardiomyocyte hypertrophy and expression of the cardio-endocrine peptide intermedin and its receptor components[J]. Cell Physiol Biochem. 2008;21(1-3):203-214.
[22] Dong F, Taylor MM, Samson WK, et al. Intermedin (adrenomedullin-2) enhances cardiac contractile function via a protein kinase C- and protein kinase A- dependent pathway in murine ventricular myocytes [J]. J Appl Physiol. 2006;101(3):778-784.
[23] Yang JH, Qi YF, Jia YX, et al. Protective effects of intermedin/adrenomedullin2 on ischemia/reperfusion injury in isolated rat hearts[J]. Peptides. 2005;26(3):501-507.
[24] Yang JH, Pan CS, Jia YX, et al. Intermedin 1-53 activates L-arginine/nitric oxide synthase/nitric oxide pathway in rat aortas[J]. Biochem Biophys Res Commun. 2006; 341(2):567-572.
[25] Chauhan M, Ross GR, Yallampalli U, et al. Adrenomedullin-2, a novel calcitonin/calcitonin-gene-related peptide family peptide, relaxes rat mesenteric artery: influence of pregnancy[J]. Endocrinology. 2007;148(4):1727-1735.
[26] Chen L, Kis B, Hashimoto H, et al. Adrenomedullin 2 protects rat cerebral endothelial cells from oxidative damage in vitro[J]. Brain Res. 2006;1086(1):42-49.
[27] Hagiwara M, Bledsoe G, Yang ZR, et al. Intermedin ameliorates vascular and renal injury by inhibition of oxidative stress[J]. Am J Physiol Renal Physiol. 2008;295(6):F1735- F1743.
[1] Koyama T, Temma K, Akera T. Reperfusion-induced contracture develops with a decreasing [Ca2+]i in single heart cells[J]. Am J Physiol. 1991;261(4 Pt 2):H1115-H1122.
[2] Basnakian AG, Ueda N, Hong X, et al. Ceramide synthase is essential for endonuclease-mediated death of renal tubular epithelial cells induced by hypoxia- reoxygenation[J]. Am J Physiol Renal Physiol. 2005;288(2):F308-F314.
[3] Combe C, Burton CJ, Dufourco P, et al. Hypoxia induces intercellular adhesion molecule-1 on cultured human tubular cells[J]. Kidney Int. 1997;51(6): 1703-1709.
[4] Doctor RB, Mandel LJ. Minimal role of xanthine oxidase and oxygen free radicals in rat renal tubular reoxygenation injury[J].J Am Soc Nephrol. 1991;1(7):959-969.
[5] Sandy Primrose, Richard Twyman, Bob Old. Principles of Gene Manipulation, Sixth Edition[M].影印版.高等教育出版社. 2002:43-85.
[6] Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP)[J]. Gene. 1996;173(1 Spec No):33-38.
[7]曹亚.实用分子生物学操作指南[M].北京:人民卫生出版社. 2003:185.
[8] Hu QH, Liang WQ. A comparative study of electroporation and iontophoresis for percutaneous penetration of naproxen[J]. Pharmazie. 2003;58(3):192-194.
[9] Jaroszeski MJ, Heller LC, Gilbert R, et al. Electrically mediated plasmid DNA delivery to solid tumors in vivo[J]. Methods Mol Biol. 2004;245:237-244.
[10] Chrenek P, Vasicek D, Makarevich AV, et al. Increased transgene integration efficiency upon microinjection of DNA into both pronuclei of rabbit embryos[J]. Transgenic Res. 2005;14(4):417-428.
[11] Koike H, Tomita N, Azuma H, et al. An efficient gene transfer method mediated by ultrasound and microbubbles into the kidney[J]. J Gene Med. 2005;7(1): 108-116.
[12] Guo DP, Li XY, Sun P, et al. Ultrasound/microbubble enhances foreign gene expression in ECV304 cells and murine myocardium[J]. Acta Biochim Biophys Sin (Shanghai). 2004;36(12):824-831.
[13] Sambrook J, Fritsch EF, Maniaatis T, et al. Molecular cloning, A laboratory mannual[M]. 2nd ed, NewYork: Cold Spring Harbor. 1995:345-347.
[14] Suqiyama M, Matsuura M, Takeuchi Y, et al. Possible mechanism of polycation liposome(PCL)-mediated gene transfer[J]. Biochim Biophys Acta. 2004;1660(1-2): 24-30.
[15] Delenda C. Lentiviral vectors: optimization of packaging, transduction and gene expression[J]. J Gene Med. 2004;6(Suppl 1):S125-S138.
[16] Ghosh SS, Gopinath P, Ramesh A. Adenoviral vectors: a promising tool for gene therapy[J]. Appl Biochem Biotechnol. 2006;133(1):9-29.
[17] Schrier RW, Wang W, Poole B, et al. Acute renal failure: definitions, diagnosis, pathogenesis, and therapy[J]. J Clin Invest. 2004; 114(1):5-14.
[18] Bonventre JV, Weinberg JM. Weinberg. Recent advances in the pathophysiology of ischemic acute renal failure[J]. J Am Soc Nephrol. 2003;14(8): 2199-2210.
[19] Chien CT, Lee PH, Chen CF,et al. De novo demonstration and co-localization of free-radical production and apoptosis formation in rat kidney subjected to ischemia/reperfusion[J]. J Am Soc Nephrol. 2001;12(5):973-982.
[20] Donnahoo KK, Shames BD, Harken AH, et al. Review article: the role of tumor necrosis factor in renal ischemia-reperfusion injury[J]. J Urol. 1999;162(1): 196-203.
[21] Yang JH, Qi YF, Jia YX, et al. Protective effects of intermedin/adrenomedullin2 on ischemia/reperfusion injury in isolated rat hearts[J]. Peptides. 2005; 26(3): 501-507.
[22] Yang JH, Jia YX, Pan CS, et al. Effects of intermedin(1-53) on cardiac function and ischemia/reperfusion injury in isolated rat hearts[J]. Biochem Biophys Res Commun. 2005;327(3):713-719.
[23] Yoshimoto T, Fukai N, Sato R, et al. Antioxidant effect of adrenomedullin on angiotensin II-induced reactive oxygen species generation in vascular smooth muscle cells[J]. Endocrinology. 2004;145(7):3331-3337.
[24] Versteilen AM, Di Maggio F, Leemreis JR, et al. Molecular mechanisms of acute renal failure following ischemia/reperfusion[J]. Int J Artif Organs. 2004;27(12): 1019-1029.
[25] Wang Q, Tang XN, Yenari MA. The inflammatory response in stroke[J]. J Neuroimmunol. 2007;184(1-2):53-68.
[26] Sharma S, Dewald O, Adrogue J, et al. Induction of antioxidant gene expression in a mouse model of ischemic cardiomyopathy is dependent on reactive oxygen species[J]. Free Radic Biol Med. 2006;40(12):2223-2231.
[27] Thurman JM. Triggers of inflammation after renal ischemia/reperfusion[J]. Clin Immunol. 2007;123(1):7-13.
[28] Halliwell B, Gutteridge JM, Cross CE. Free radicals, antioxidants, and human disease: where are we now?[J]. J Lab Clin Med. 1992;119(6):598-620.
[29]陈瑗,周玫.自由基医学基础与病理生理[M].北京:人民卫生出版社. 2002:3-52.
[30] Chen L, Kis B, Hashimoto H, et al. Adrenomedullin 2 protects rat cerebral endothelial cells from oxidative damage in vitro[J]. Brain Res. 2006;1086(1): 42-49.
[31] Nogueira-Machado JA, Lima e Silva FC, Medina LO, et al. Modulation of the reactiveoxygen species (ROS) generation mediated by cyclic AMP-elevating agents or Interleukin 10 in granulocytes from type 2 diabetic patients (NIDDM): a PKA-independent phenomenon[J]. Diabetes Metab. 2003;29(5):533-537.
[1] Wimalawansa SJ. Amylin, calcitonin gene-related peptide, calcitonin, and adrenomedullin: a peptide superfamily[J]. Crit Rev Neurobiol. 1997;11(2-3): 167-239.
[2] Roh J, Chang CL, Bhalla A, et al. Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complexes[J]. J Biol Chem. 2004;279(8):7264-7274.
[3] Takei Y, Inoue K, Ogoshi M, et al. Identification of novel adrenomedullin in mammals: a potent cardiovascular and renal regulator[J]. FEBS Lett. 2004;556 (1-3):53-58.
[4] Yang JH, Jia YX, Pan CS, et al. Effects of intermedin(1-53) on cardiac function and ischemia/reperfusion injury in isolated rat hearts[J]. Biochem Biophys Res Commun. 2005;327(3):713-719.
[5] Chang CL, Roh J, Hsu SY. Intermedin, a novel calcitonin family peptide that exists in teleosts as well as in mammals: a comparison with other calcitonin/ intermedin family peptides in vertebrates[J]. Peptides. 2004;25(10): 1633-1642.
[6] Poyner DR, Sexton PM, Marshall I, et al. International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors[J]. Pharmacol Rev. 2002;54(2):233-246.
[7] Kindt F, Wiegand S, Loser C, et al. Intermedin: a skin peptide that is downregulated in atopic dermatitis[J]. J Invest Dermatol. 2007;127(3):605-613.
[8] Takahashi K, Kikuchi K, Maruyama Y, et al. Immunocytochemical localization of adrenomedullin 2/intermedin-like immunoreactivity in human hypothalamus, heart and kidney[J]. Peptides. 2006;27(6):1383-1389.
[9] Takei Y, Hyodo S, Katafuchi T, et al. Novel fish-derived adrenomedullin in mammals: structure and possible function[J]. Peptides. 2004;25(10):1643-1656.
[10] Pan CS, Yang JH, Cai DY, et al. Cardiovascular effects of newly discovered peptide intermedin/adrenomedullin-2[J]. Peptides. 2005;26(9):1640-1646.
[11] Zhao Y, Bell D, Smith LR, et al. Differential effects of components of the cardiomyocyte adrenomedullin/intermedin receptor system following blood pressure reduction in nitric oxide-deficient hypertension[J]. J Pharmacol Exp Ther. 2006;316(3): 1269-1281.
[12] Morimoto R, Satoh F, Murakami O, et al. Expression of adrenomedullin2/ intermedin in human brain, heart, and kidney[J]. Peptides. 2007;28(5):1095-1103.
[13] McLatchie LM, Fraser NJ, Main MJ, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor–like receptor[J]. Nature. 1998;393 (6683): 333-339.
[14] Yang JH, Qi YF, Jia YX, et al. Protective effect s of intermedin/adrenomedullin2 on ischemia/ reperfusion injury in isolated rat hearts[J]. Peptides. 2005;26(3): 501-507.
[15] Jia YX, Yang JH, Pan CS, et al. Intermedin1-53 protects the heart against isoproterenol-induced ischemic injury in rats[J]. Eur J Pharmacol. 2006;549(1-3): 117-123.
[16] Dong F, Taylor MM, Samson WK, et al. Intermedin (adrenomedullin-2) enhances cardiac contractile function via a protein kinase C- and protein kinase A- dependent pathway in murine ventricular myocytes [J]. J Appl Physiol. 2006;101(3):778-784.
[17] Beltowski J, Jamroz A. Adrenomedullin-what do we know 10 years since its discovery[J]?Pol J Pharmacol. 2004;56(1):5-27.
[18] Terata K, Miura H, Liu Y, et al. Human coronary arteriolar dilation to adrenomedullin: role of nitric oxide and K(+) channels[J]. Am J Physiol Heart Circ Physiol. 2000;279(6): H2620-H2626.
[19] Sata M, Kakoki M, Nagata D, et al. Adrenomedullin and nitric oxide inhibit human endothelial cell apoptosis via acyclic GMP2 independent mechanism[J]. Hypertension. 2000;36(1):83-88.
[20] Yang JH, Pan CS, Jia YX, et al. Intermedin 1-53 activates L-arginine/nitric oxide synthase/nitric oxide pathway in rat aortas[J]. Biochem Biophys Res Commun. 2006;341(2):567-572.
[21] Chauhan M, Ross GR, Yallampalli U, et al. Adrenomedullin-2, a novel calcitonin/calcitonin-gene-related peptide family peptide, relaxes rat mesenteric artery: influence of pregnancy[J]. Endocrinology. 2007;148(4):1727-1735.
[22] Kandilci HB, Gumusel B, Lippton H. Intermedin/adrenomedullin-2 (IMD/AM2) relaxes rat main pulmonary arterial rings via cGMP-dependent pathway: role of nitric oxide and large conductance calcium-activated potassium channels (BK(Ca))[J]. Peptides. 2008;29 (8):1321-1328.
[23] Taylor MM, Bagley SL, Samson WK. Intermedin/adrenomedullin-2 acts within central nervous system to elevate blood pressure and inhibit food and water intake[J]. Am J Physiol Regul Integr Comp Physiol. 2005;288(4):R919-R927.
[24] Ren YS, Yang JH, Zhang J, et al. Intermedin 1-53 in central nervous system elevates arterial blood pressure in rats[J]. Peptides. 2006;27(1):74-79.
[25] Cui H, Kohsaka A, Waki H, et al. Adrenomedullin 2 microinjection into the nucleus tractus solitarius elevates arterial pressure and heart rate in rats[J]. Auton Neurosci. 2008;142(1-2):45-50.
[26] Charles CJ, Rademaker MT, Richards AM. Hemodynamic, hormonal, and renal actions ofadrenomedullin-2 in normal conscious sheep[J]. Endocrinology. 2006; 147(4):1871-1877.
[27] Abdelrahman AM, Pang CC. Effect of intermedin/adrenomedullin-2 on venous tone in conscious rats[J]. Naunyn Schmiedebergs Arch Pharmacol. 2006;373(5): 376-380.
[28] Fujisawa Y, Nagai Y, Miyatake A, et al. Renal effects of a new member of adrenomedullin family, adrenomedullin2, in rats[J]. Eur J Pharmacol. 2004;497(1): 75-80.
[29] Fujisawa Y, Nagai Y, Miyatake A, et al. Roles of adrenomedullin2 in regulating the cardiovascular and sympathetic nervous systems in conscious rats[J]. Am J Physiol Heart Circ Physiol. 2006;59(3):H1120-H1127.
[30] Fujisawa Y, Nagai Y, Miyatake A, et al. Effects of adrenomedullin2 on regional hemodynamics in conscious rats[J]. Eur J Pharmacol. 2007;558(1-3):128-132.
[31]王曦,曾强,袁鹰,等.肾上腺髓质素2对自发性高血压大鼠血压和心功能的影响[J].中华高血压杂志. 2007;15(12):1012-1016.
[32] Kobayashi Y, Liu YJ, Gonda T, et al. Coronary vasodilatory response to a novel peptide, adrenomedullin 2[J]. Clin Exp Pharmacol Physiol. 2004:31(Suppl 2):S49-S50.
[33] Doganci S, Yildirim V, Bolcal C, et al. Intermedin (IMD/AM2) dilates the pig coronary vascular bed through release of nitric oxide[J]. FASEB J. 2007:21(6): 1380-1385.
[34] Bell D, Zhao Y, McCoy FGP, et al. Differential effects of an anti-oxidant intervention on cardiomyocyte expression of adrenomedullin and intermedin and their receptor components in chronic nitric oxide deficiency[J]. Cell Physiol Biochem.2007;20(5): 269-282.
[35] Bell D, Zhao Y, Devine AB, et al. Influence of atenolol and nifedipine on nitric-oxide deficient cardiomyocyte hypertrophy and expression of the cardio-endocrine peptide intermedin and its receptor components[J]. Cell Physiol Biochem. 2008;21(1-3):203-214.
[36] Bell D, Zhao Y, McCoy FP, et al. Expression of the counter-regulatory peptide intermedin is augmented in the presence of oxidative stress in hypertrophied cardiomyocytes[J]. Cell Physiol Biochem. 2008;21(5-6):409-420.
[37] Zeng Q, Yuan Y, Wang X, et al. Upregulated expression of intermedin and its receptor in the myocardium and aorta in spontaneously hypertensive rats[J]. Peptides. 2009;30(2): 391-399.
[38] Hirose T, Totsune K, Mori N, et al. Increased expression of adrenomedullin 2/intermedin in rat hearts with congestive heart failure[J]. Eur J Heart Fail. 2008;10(9):840-849.
[39]张玉青,黄卡特,毛孙忠,等.高脂血症和高脂血症性脂肪肝大鼠肾上腺髓质素2的变化[J].世界华人消化杂志. 2007;15(21):2341-2344.
[40]陈斌,石洪波,张雪军,等. Intermedin预处理对大鼠肾缺血离体再灌注损伤的保护作用[J].临床泌尿外科杂志. 2008;23(5):385-388.
[41] Hagiwara M, Bledsoe G, Yang ZR, et al. Intermedin ameliorates vascular and renal injury by inhibition of oxidative stress[J]. Am J Physiol Renal Physiol. 2008;295(6): F1735-F1743.
[42] Burak Kandilci H, Gumusel B, Wasserman A, et al. Intermedin/adrenomedullin-2 dilates the rat pulmonary vascular bed: dependence on CGRP receptors and nitric oxide release[J]. Peptides. 2006;27(6):1390-1396.
[43]于晓敏,刘新民,齐永芬,等.中介素1-53对油酸致大鼠急性肺损伤的保护作用[J].中华结核和呼吸杂志. 2006;29(12):808-811.
[44]于晓敏,刘新民,齐永芬,等.中介素及其受体系统在油酸致大鼠急性肺损伤中的变化和意义[J].北京大学学报(医学版). 2006;38(5):496-500.
[45] Gong YS, Fan XF, Wu XM, et al. Changes of intermedin/adrenomedullin 2 and its receptors in the right ventricle of rats with chronic hypoxic pulmonary hypertension[J]. Sheng Li Xue Bao. 2007;59(2):210-214.
[46]刘青,王新均,董有平,等.败血症休克大鼠肺组织Intermedin含量及其mRNA表达[J].郧阳医学院学报. 2007;26(2):74-77.
[47]吴东红,田丽鲜,范小芳,等.低氧对大鼠肺组织中介素及其受体系统表达的影响[J].中国微循环. 2007;11(2):94-97.
[48]范小芳,黄萍,龚永生,等.慢性低氧性肺动脉高压大鼠肺组织肾上腺髓质素2的变化[J].中国应用生理学杂志. 2007;23(4):467-471.
[49] Hashimoto H, Hyodo S, Kawasaki M, et al. Centrally administered adrenomedullin 2 activates hypothalamic oxytocin-secreting neurons, causing elevated plasma oxytocin level in rats[J]. Am J Physiol Endocrinol Metab. 2005;289(5):E753-E761.
[50] Hashimoto H, Hyodo S, Kawasaki M, et al. Adrenomedullin 2 (AM2)/intermedin is a more potent activator of hypothalamic oxytocin-secreting neurons than AM possibly through an unidentified receptor in rats. Peptides[J]. 2007;28(5): 1104-1012.
[51] Taylor MM, Samson WK. Stress hormone secretion is altered by central administration of intermedin/adrenomedullin-2[J]. Brain Res. 2005;1045(1-2): 199-205.
[52] Taylor MM, Bagley SL, Samson WK. Intermedin/Adrenomedullin-2 inhibits growth hormone release from cultured, primary anterior pituitary cells[J]. Endocrinology. 2006;147(2):859-864.
[53] Lin Chang C, Roh J, Park JI, et al. Intermedin functions as a pituitary paracrine factor regulating prolactin release[J]. Mol Endocrinol. 2005;19(11):2824-2838.
[54] Chen L, Kis B, Hashimoto H, et al. Adrenomedullin 2 protects rat cerebral endothelial cellsfrom oxidative damage in vitro[J]. Brain Res. 2006;1086(1): 42-49.
[55]蔡莉蓉,李玉珍,刘秀华.肾上腺髓质素2对大鼠脑微血管内皮细胞增殖的影响[J].中国病理生理杂志. 2006;22(5):908-910.
[56] White MM, Samson WK. Intermedin 17-47 does not function as a full intermedin antagonist within the central nervous system or pituitary[J]. Peptides. 2007;28(11): 2171-2178.
[57] Owji AA, Chabot JG, Dumont Y, et al. Adrenomedullin-2/intermedin induces cAMP accumulation in dissociated rat spinal cord cells: evidence for the existence of a distinct class of sites of action[J]. J Mol Neurosci. 2008;35(3):355-361.
[58] Nishikimi T. Adrenomedullin in the kidney-renal physiological and pathophysiological roles[J]. Curr Med Chem. 2007;14(15):1689-1699.
[59] Nishimatsu H, Hirata Y, Shindo T, et al. Role of endogenous adrenomedullin in the regulation of vascular tone and ischemic renal injury: studies on transgenic/knockout mice of adrenomedullin gene[J]. Circ Res. 2002;90(6): 657-663.
[60] Chao J, Kato K, Zhang JJ, et al. Human adrenomedullin gene delivery protects against cardiovascular remodeling and renal injury[J]. Peptides. 2001;22(11): 1731-1737.