【作者】 金秀男；

【导师】 崔勋；

【作者基本信息】 延边大学 ， 生理学， 2011， 博士

【摘要】 肾缺血再灌注损伤(ischemia reperfusion injury, IRI)是肾组织缺血时和其后恢复血液灌注时器官功能不能恢复正常,甚至发生更为严重的组织损伤或器官功能衰竭。肾脏由于其组织结构和功能的特殊性,对缺血再灌注损伤特别敏感,临床上常见因缺血引起急性肾功能衰竭(acute renal failue, ARF)的发生,如急性缺血性肾功衰竭、心肺复苏、心脏体外循环、失血性休克、严重脱水、弥散性血管内凝血、心脏手术及肾脏移植术等过程中,特别是多器官功能衰竭的危重患者几乎都伴有ARF的发生。尽管进行积极的对症支持治疗,但还是出现很高的发病率和死亡率。实验证明,心房钠尿肽(atrial natriuretic peptide, ANP)可有效地减轻心脏、肝脏及胃等器官的缺血再灌注损伤,起明显的保护作用。近年来研究表明,ANP对大鼠肾缺血再灌注损伤也具有保护作用,其保护作用是增加肾小球滤过率(glomerular filtration rate, GFR)和肾髓质的血流量,减轻髓质部生成表皮生长因子(epithelial growth factor, EGF)细胞的损伤,恢复肾小管上皮细胞的完整性；ANP可减轻中性白细胞的活性,抑制其释放各种炎症介质和氧自由基的生成保护肾功能,但其作用机制尚不清楚。钠尿肽(natriuretic peptides, NPs)是新发现的肽类激素,主要有ANP、脑钠尿肽(brain natriuretic peptide, BNP)及C-型钠尿肽(C-type natriuretic peptide,CNP)等,其共有的效应是利尿、排钠、舒张血管、降低血压和调节水电解质平衡。ANP和BNP主要由心脏合成和分泌,而CNP主要由内皮细胞分泌,是脑或神经系统特异性表达的神经递质。现已确认能与钠尿肽结合的受体有三种亚型：A型钠尿肽受体(natriuretic peptide receptor type A, NPR-A)、B型钠尿肽受体(natriuretic peptide receptor type B, NPR-B)及清除型钠尿肽受体(natriuretic peptide clearance receptor, NPR-C)。ANP和BNP主要与NPR-A结合,CNP则主要与NPR-B结合而发挥其生物学效应。研究表明,钠尿肽及其受体广泛分布在肾脏,但是NPs-NPR-cGMP系统在肾缺血再灌注的病理生理学作用尚不甚清楚。为此,本研究从形态学和分子生物学等角度观察和探讨CNP-NPR-B-cGMP信号通路在肾缺血再灌注损伤中的作用及其机制。本研究利用大鼠肾缺血再灌注模型,制备血清样本测定肌酐(creatinine, Cr)、尿素氮(blood urine nitrogen, BUN)等肾功能指标,且利用光镜和电镜观察肾组织形态学及肾组织超微结构,利用放射免疫分析和酶联免疫法测定血清CNP浓度,采用RT-PCR和免疫组织化学方法观察CNP及NPR-B mRNA的表达和分布。另外,取24只SD大鼠随机分为假手术组(sham)、肾缺血再灌注组(ischemia reperfusion, IR)及肾缺血再灌注即时CNP给药组(IR+CNP)(每组8只),测定血清Cr、BUN及肾组织SOD活性及MDA含量,且利用光镜观察肾组织形态学变化和采用免疫印迹技术观察肾组织Bax、Bcl-2的表达变化。实验结果如下：1.大鼠肾缺血再灌注组血清Cr、BUN水平且明显高于假手术组(n=8,P<0.01),并呈现时间依赖性特征。2.假手术组肾脏组织结构清楚,肾小管上皮细胞完整,只有轻度炎性细胞浸润；IR组肉眼观察可见肾脏体积明显增大而质软,剖面见肾髓质呈暗红色；皮质肿胀而苍白。显微镜下观察,IR组近端肾小管上皮细胞空泡及颗粒变性,细胞扁平,管腔扩张,刷状缘脱落消失,管腔内有脱落的上皮、管型和炎症渗出物；基底膜裸露,甚至呈现肾小管细胞的节段性灶状坏死。肾小管间质有不同程度的炎症细胞浸润和水肿。远端肾小管和集合管管腔内可见细胞碎片或颗粒管型,肾小管基底膜节段性断裂,断裂部位可见淋巴细胞、单核细胞及中性粒细胞浸润,肾间质弥漫并水肿。sham组电镜下超微结构基本正常,线粒体嵴完整,未见肿胀,粗面内质网结构完整,排列整齐；IR组电镜下可见肾小管上皮细胞微绒毛脱落,线粒体肿胀、线粒体嵴消失,内质网扩张,初级和次级溶酶体增多,吞噬空胞亦可见有增多现象。甚至上皮细胞崩解脱落,仅可见裸露或断裂的基底膜。3.假手术组及缺血再灌注组不同时间段(0/1/2/4h)血清CNP含量无明显差异(n=8,P>0.05)。4.缺血再灌注组肾组织CNP mRNA表达明显上调,且在再灌注2 hour时表达最高(n=8,P<0.05)。5.假手术组及缺血再灌注组肾组织NPR-B均呈阳性,可见到散在分布的棕色颗粒,但与假手术组相比再灌注组呈阳性的棕色颗粒明显增多,染色明显加深。另外,缺血再灌注组大鼠肾组织中NPR-B mRNA表达明显高于正常对照组。6.假手术组肾脏组织结构清楚,肾小管上皮细胞完整,只有轻度炎性细胞浸润；再灌注组肾脏体积明显增大而质软,剖面见肾髓质呈暗红色；皮质肿胀而苍白。显微镜下近端肾小管上皮细胞空泡及颗粒变性,细胞扁平,管腔扩张,刷状缘脱落消失,管腔内有脱落的上皮、管型和炎症渗出物,基底膜裸露,甚至可见肾小管细胞的节段性灶状坏死。肾小管间质可有不同程度的炎症细胞浸润和水肿,远端肾小管和集合管管腔内可见细胞碎片或颗粒管型。肾小管基底膜节段性断裂,断裂部位可见淋巴细胞、单核细胞及中性粒细胞浸润,肾间质弥漫水肿。肾小球和肾小动脉未见明显改变。CNP组肾小管排列基本正常,以肿胀为主,肾间质水肿、充血,炎性细胞浸润不明显。7.缺血再灌注组24小时后血清Cr、BUN水平明显高于假手术组(n=8,P<0.001);CNP即时给药组较缺血再灌注组明显下降(n=8,P<0.05)。8.缺血再灌注组与假手术组相比MDA含量显著升高,SOD活性显著下降(n=8,P<0.01和P<0.05)；CNP即时给药组与IR组相比MDA含量明显减少,SOD活性有所提高(n=8,P<0.01和P<0.05)。9.与假手术组及CNP即时给药组相比,缺血再灌注组肾组织Bax表达明显增加(n=8,P<0.05),而Bcl-2的表达在3组之间无明显差异,但是CNP即时给药组Bax/Bcl-2比值降低(n=8,P<0.05)。以上结果提示：1.肾缺血再灌注时组织结构和功能明显受到损伤,尤其在皮髓交界区和髓质部位。2.肾缺血再灌注损伤时肾组织CNP及NPR-B的表达明显上调。3.CNP明显改善缺血再灌注损伤的肾脏组织结构和功能,呈现抗损伤作用。4.CNP对损伤的肾组织具有明显的抗氧化作用。5.CNP对缺血再灌注肾具有明显的抗细胞凋亡效应,其作用是通过上调抗凋亡基因表达的途径实现的。更多还原

【Abstract】 Ischemia reperfusion (IR) may caused renal structural and functional injury in kidney. Because of the renal particularity of structure and function, kidney was showed especially sensitive for ischemia reperfusion injury (IRI). Therefore, there were common renal diseases in clinical patients such as acute renal failure (ARF) and showed high morbidity and mortality in patients. Experimental data demonstrated that atrial natriuretic peptide (ANP)may reduced reperfusion injuries of the heart, liver, and the stomach etc and showed evident protective effcts in human and several animals. Is was also demonstrated that ANP may had protective function in IRI rat kidney via increase renal glomerular filtration rate, blood flow in medullar and reduce of epithelial cells injury in renal medullar and therefore recuperates integrality of dubular epithelial cells. On the other hand, ANP my reduces neutrophilic activity, inhibits secreiton of mediators of inflammations and reduces production of reactive oxygen species. However, the mechanism of ANP on ischemia reperfusion injury especially for ARF is not well kown.Natriuretic peptides (NPs), as a family of hormen, thare are four members in NPs I. E. ANP, brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). NPs may induce diuresis, sodium excretion, vessel dilatation, blood pressure regulation and electrolyte balance. The heart sythesis and release of ANP and BNP. CNP is sythesised by endothelial cells and play paracrine function in serise of tissues, such as the heart, vessel, nervous system etc. There are three types of the NP receptors (NPRs), NPR-A, NPR-B and NPR-C (NP clearance receptor). ANP and BNP mainly bingding to NPR-A and CNP maily bingding to NPR-B. Several experiments indicate that NPs and NPRs distributed in kidney but effects of NPs-cGMP on renal ischemia reperfusion injury are not clear. Therefor, the purpose of the precent is to investigate the effect of CNP-NPR-B-cGMP signaling pathway on renal ischemia reperfusion injury by using rat renal ischemia reperfusion model. immunohistochemistry methods, RT-PCR, ELISA etc. The renal structural changes are observed by microscope. Renal superoxide dismutase (SOD) activities, malondialdehyde (MDA) levels, Bax and Bcl-2 gene expressions are also to detected. In addition, creatinine (Cr) and blood urine nitrogen (BUN) levels are measured by autobiochemicaldetector. On the other hand, the rats will be randomly divided into three groups:sham operration group(sham), IR group(IR)and IR+CNP group (IR+CNP) (n=8 in each groups).The results of the precent experiments as follows:1. Levels of Cr and BUN were increaseed by time-dependent manna after renal ischemia reperfusion (n=8, P<0.01 vs sham goup).2. In sham group, renal organization was clearly and epithelial cells of tubules were completed, only a minor inflammatory cells were observed. In IR group, under microscope can be seen near the end of the epithelial cells from an empty bubble and particles of tunica adventitia. The flat, the paint off, or fate, the detachment of the epithelial cells, inflammatory exudate. The basement membrane is bare. Glomerular and kidney arterioles not change. sham group the structure of under electronic microscope is normal, mitochondria integrity, not swell, rough endoplasmic reticulum structural integrity; and IR group the nephridial tube epithelial cells microvilli off, mitochondria turgescence, mitochondria and endoplasmic abnormal reticulum distention, primary and secondary mixed up in a body and empty cell can also see that there is increasing. even the epithelial cells from collapse, and only thus is bare or break the substrate.3. The levels of CNP were unchanged in sham and IR groups (n=8, P>0.05 vs sham group).4. Expression of CNP mRNA were increased in IR group rats and showed time-dependent manna (n=8, P<0.05). The peake of CNP mRNA expression were observed at 2 houres after perfused in rats.5. In sham group, immunohistochemical data demonstrated that NPR-B were distributed widly in kidney. In IR group, renal NPR-B were increased compared with sham group significiently. RT-PCR data of NPR-B were similarly about with the immunohistochemical data.6. After RIR the histological morphologh analysis demonstrated phathologic change significantly. The electron microscope data indicated that IR caused mitochondrial injury and showed obviously swelling, some of the cristae became vacular-like; the microvilli of the Proximal tubule cells were scare and swelled, rough endoplasmic reticulums were reduced in number, foamy changes in the structure of nuclear and cytoplasm were also observed. In CNP group, the microvilli were slightly swelled or kept intact; the cristae of the mitochondria were slightly changed but the structure was still intact; rough endoplasmic reticulums were slightly swelled ribsome was all preserved, nuclear had almost normal figure with very clear nucleolus.7. Serum levels of Cr and BUN in IR group rats were significantly increased after reperfused for 24 hours (n=8, P<0.001vs sham group), and the serum levels of Cr and BUN were significantly reduced by CNP pretreatment after renal ischemia reperfusion (n=8, P<0.05 vs IR group).8. In IR group, the MDA contents were increased and SOD activity were significantly reduced after renal reperfusion (n=8, P<0.001and P<0.05 vs sham group). CNP may significantly reduced levels of MDA and slightly increased the activity of SOD after ischemia reperfusion in rat kidney (n=8, P<0.001and P<0.05 vs sham group).9. Reanal Bax gene expression was increased by ischemia reperfusion injury (n=8, P<0.05 vs sham or IR+CNP groups) without changes of Bcl-2 expression, but the ratio of Bax/Bcl-2 was decreased by CNP treatment in IR+CNP group (n=8, P<0.05 vs IR group).Thease results indicat that:1. Ischemia reperfusion my causes renal structural and functional injury especially in renal junctional zone of the cortex and medulla in rat kidney.2. Renal ischemia reperfusion injury induces up rangulation of CNP and NPR-B mRNA expression in rat kidney.3. CNP may improves renal structrue and function in ischemia reperfusion injury rat kidney.4. CNP has been shown antioxidant effect in rat kidney.5. CNP has been shown antiapoptosis effect in ischemia reperfusion rat kidney via up regulation of Bax/Bcl-2 ratio pathway.更多还原