CNP-NPR-B-cGMP信号通路在肾缺血再灌注损伤中的作用
发布时间：2018-10-11 08:22
【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,P0.01),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,P0.05)。 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,P0.05)。 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,P0.001);CNP即时给药组较缺血再灌注组明显下降(n=8,P0.05)。 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,P0.01和P0.05)；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,P0.01和P0.05)。 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,P0.05),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,P0.05)。 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。
[Abstract]:Renal ischemia-reperfusion injury (IRI) is the function of organ failure in renal tissue ischemia and later recovery of blood perfusion, and even more serious tissue injury or organ failure. Due to the particularity of its structure and function, the kidney is particularly sensitive to ischemia-reperfusion injury. In clinic, acute renal failure caused by ischemia, such as acute ischemic renal failure, cardiopulmonary resuscitation, cardiopulmonary bypass, hemorrhagic shock, Severe dehydration, disseminated intravascular coagulation, cardiac surgery, and kidney transplantation, especially in critically ill patients with multiple organ failure, are almost accompanied by the occurrence of death. Despite positive symptomatic support, there was still a high incidence of morbidity and mortality. The experimental results show that atrial natriuretic peptide (ANP) can effectively reduce the ischemia-reperfusion injury of heart, liver and stomach. Recent studies have shown that ANP has a protective effect on renal ischemia-reperfusion injury in rats, and its protective effect is to increase the glomerular filtration rate (GFR) and the blood flow of renal medulla, and to reduce the damage of epidermal growth factor (EGF) cells in medulla. To restore the integrity of renal tubular epithelial cells, ANP can reduce the activity of neutrophils and inhibit the release of various inflammatory mediators and oxygen free radicals to protect renal function, but its mechanism of action is not clear. The natriuretic peptide (NPs) is a newly discovered peptide hormone, mainly including ANP, brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). The common effect is diuretic, sodium excretion, vasodilation, blood pressure reduction and water electrolyte regulation. The balance. ANP and BNP are mainly synthesized and secreted by the heart, while CNP is mainly secreted by endothelial cells, which is the nerve specifically expressed by the brain or nervous system. It has been recognized that there are three subtypes of receptors capable of binding to natriuretic peptide: Atype natriuretic peptide receptor type A (NPR-A), type B natriuretic peptide receptor type B (NPR-B) and clear natriuretic peptide receptor (NPR-C). ANP and BNP are mainly combined with NPR-A, and CNP is mainly combined with NPR-B to exert its biology. The results show that the natriuretic peptide and its receptors are widely distributed in the kidney, but the pathophysiological role of NPs-NPR-cGMP system in renal ischemia-reperfusion is not very important. To this end, we observed and discussed the effect of CNP-NPR-B-cGMP signal pathway in renal ischemia-reperfusion injury from the aspects of morphology and molecular biology. Using the model of renal ischemia-reperfusion in rats, serum samples were prepared to determine renal function indices such as creatidine, Cr, blood urine, and so on, and renal tissue morphology was observed by light microscope and electron microscope. The concentrations of CNP and NPR-B mR were observed by RT-PCR and immunohistochemistry. In addition, 24 SD rats were randomly divided into sham operation group (sham), renal ischemia reperfusion group (IR) and renal ischemia-reperfusion instant CNP administration group (IR + CNP) (8 rats in each group). Serum Cr, NPY and SOD in renal tissues were determined. The activity and MDA content were observed, and the morphological changes of renal tissues were observed by light microscope and the expression of Bax and Bc in renal tissues were observed by western blot technique. expression change of l-2 The results of the experiment were as follows: 1. The levels of serum Cr and NPY in renal ischemic reperfusion group were significantly higher than that in sham operation group (n = 8, P <0.01). 01) Time-dependent characteristics were presented and time-dependent characteristics were presented. 2. The renal tissue structure of the sham-operated group was clear, the renal tubular epithelial cells were intact, only mild inflammatory cell infiltration was observed. It was seen that the renal medulla was dark red; the cortex was swollen and pale. Under the microscope, the vacuoles and degeneration of the proximal tubular epithelial cells of the IR group were observed, the cells were flat, the lumen of the tube was expanded, the brush-like margin disappeared, and the epithelial, tubular and inflammatory exudates were removed in the lumen of the tube. The basement membrane was exposed. Even the segmental focal necrosis of the renal tubular cells. There is a different degree of inflammatory cell infiltration and edema in the stroma. The distal tubules and the lumen of the collection tube are visible cell debris or particle casts, tubular basement membrane segment fracture, fracture site visible lymphocytes, monocytes Neutrophil infiltration, renal interstitial diffusion and edema were observed. The ultrastructures under electron microscope of sham group were basically normal, the mitochondria were intact, swelling was not observed, the structure of rough reticulum was complete, the arrangement was orderly, and the microvilli of renal tubular epithelial cells showed off in the IR group under electron microscope. Swelling of granule, disappearance of mitochondrial membrane, dilatation of ER, primary and secondary dissolution There is an increase in the number of enzymes, and an increase in the number of cells can be seen. Even the epithelial cells Disintegration, only visible or broken basal membrane. 3. sham operation group and ischemia-reperfusion group for different time periods (0/ 1/ 2/ 4h) serum C There was no significant difference in NP content (n = 8, P0.05). The expression of NPR-B was highest (n = 8, P 0.05). 5. Both the sham operation group and the renal tissue NPR-B of the ischemia-reperfusion group were positive. Compared with the reperfusion group, the positive brown granules increased significantly, and the staining was significantly increased. The expression of NPR-B mRNA in renal tissues of rats was significantly higher than that in normal control group. The dirty volume is obviously increased and the quality is soft; the section is seen in the medulla of the kidney; the renal medulla is dark red; the cortex is swollen and pale. The near-end tubular epithelial cell vacuoles and particle degeneration under the microscope, the cell is flat, the tube cavity is expanded, the brush-like edge falls off, and the exfoliated epithelium, the tube and the tube cavity are formed in the tube cavity. Inflammatory exudate, bare basement membrane, and even segmental focal necrosis of tubular cells. Renal tubular stroma can have varying degrees of inflammatory cells. Infiltration and edema, visible cell debris or particle casts in the distal tubules and manifold lumen. Tubular basement membrane segment fracture, fracture Site visible lymphocytes, monocytes, and neutralizing Neutrophil infiltration, renal interstitial diffuse edema. No significant changes in glomerular and renal function were found. Renal tubular arrangement of CNP group After 24 hours of ischemia-reperfusion group, the level of serum Cr and NPY was significantly higher than that of sham-operated group (n = 8, P0.001). (n = 8, P0.05). Compared with the sham operation group, the content of MDA increased significantly and the activity of SOD decreased significantly (n = 8, P0.01 and P0.05). Compared with sham operation group and CNP, the expression of Bax was significantly increased (n = 8, P0.05). Significant differences, but C The ratio of Bax/ Bcl-2 decreased (n = 8, P0.05). Results: 1. The structure and function of tissue were obviously damaged during renal ischemia-reperfusion. especially at the junctional region and medulla of the skin. The expression of NPR-B was obviously up-regulated. To improve the structure and function of kidney tissue damaged by ischemia-reperfusion injury, and present an anti-injury effect.
【学位授予单位】：延边大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
本文编号：2263501
[Abstract]:Renal ischemia-reperfusion injury (IRI) is the function of organ failure in renal tissue ischemia and later recovery of blood perfusion, and even more serious tissue injury or organ failure. Due to the particularity of its structure and function, the kidney is particularly sensitive to ischemia-reperfusion injury. In clinic, acute renal failure caused by ischemia, such as acute ischemic renal failure, cardiopulmonary resuscitation, cardiopulmonary bypass, hemorrhagic shock, Severe dehydration, disseminated intravascular coagulation, cardiac surgery, and kidney transplantation, especially in critically ill patients with multiple organ failure, are almost accompanied by the occurrence of death. Despite positive symptomatic support, there was still a high incidence of morbidity and mortality. The experimental results show that atrial natriuretic peptide (ANP) can effectively reduce the ischemia-reperfusion injury of heart, liver and stomach. Recent studies have shown that ANP has a protective effect on renal ischemia-reperfusion injury in rats, and its protective effect is to increase the glomerular filtration rate (GFR) and the blood flow of renal medulla, and to reduce the damage of epidermal growth factor (EGF) cells in medulla. To restore the integrity of renal tubular epithelial cells, ANP can reduce the activity of neutrophils and inhibit the release of various inflammatory mediators and oxygen free radicals to protect renal function, but its mechanism of action is not clear. The natriuretic peptide (NPs) is a newly discovered peptide hormone, mainly including ANP, brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). The common effect is diuretic, sodium excretion, vasodilation, blood pressure reduction and water electrolyte regulation. The balance. ANP and BNP are mainly synthesized and secreted by the heart, while CNP is mainly secreted by endothelial cells, which is the nerve specifically expressed by the brain or nervous system. It has been recognized that there are three subtypes of receptors capable of binding to natriuretic peptide: Atype natriuretic peptide receptor type A (NPR-A), type B natriuretic peptide receptor type B (NPR-B) and clear natriuretic peptide receptor (NPR-C). ANP and BNP are mainly combined with NPR-A, and CNP is mainly combined with NPR-B to exert its biology. The results show that the natriuretic peptide and its receptors are widely distributed in the kidney, but the pathophysiological role of NPs-NPR-cGMP system in renal ischemia-reperfusion is not very important. To this end, we observed and discussed the effect of CNP-NPR-B-cGMP signal pathway in renal ischemia-reperfusion injury from the aspects of morphology and molecular biology. Using the model of renal ischemia-reperfusion in rats, serum samples were prepared to determine renal function indices such as creatidine, Cr, blood urine, and so on, and renal tissue morphology was observed by light microscope and electron microscope. The concentrations of CNP and NPR-B mR were observed by RT-PCR and immunohistochemistry. In addition, 24 SD rats were randomly divided into sham operation group (sham), renal ischemia reperfusion group (IR) and renal ischemia-reperfusion instant CNP administration group (IR + CNP) (8 rats in each group). Serum Cr, NPY and SOD in renal tissues were determined. The activity and MDA content were observed, and the morphological changes of renal tissues were observed by light microscope and the expression of Bax and Bc in renal tissues were observed by western blot technique. expression change of l-2 The results of the experiment were as follows: 1. The levels of serum Cr and NPY in renal ischemic reperfusion group were significantly higher than that in sham operation group (n = 8, P <0.01). 01) Time-dependent characteristics were presented and time-dependent characteristics were presented. 2. The renal tissue structure of the sham-operated group was clear, the renal tubular epithelial cells were intact, only mild inflammatory cell infiltration was observed. It was seen that the renal medulla was dark red; the cortex was swollen and pale. Under the microscope, the vacuoles and degeneration of the proximal tubular epithelial cells of the IR group were observed, the cells were flat, the lumen of the tube was expanded, the brush-like margin disappeared, and the epithelial, tubular and inflammatory exudates were removed in the lumen of the tube. The basement membrane was exposed. Even the segmental focal necrosis of the renal tubular cells. There is a different degree of inflammatory cell infiltration and edema in the stroma. The distal tubules and the lumen of the collection tube are visible cell debris or particle casts, tubular basement membrane segment fracture, fracture site visible lymphocytes, monocytes Neutrophil infiltration, renal interstitial diffusion and edema were observed. The ultrastructures under electron microscope of sham group were basically normal, the mitochondria were intact, swelling was not observed, the structure of rough reticulum was complete, the arrangement was orderly, and the microvilli of renal tubular epithelial cells showed off in the IR group under electron microscope. Swelling of granule, disappearance of mitochondrial membrane, dilatation of ER, primary and secondary dissolution There is an increase in the number of enzymes, and an increase in the number of cells can be seen. Even the epithelial cells Disintegration, only visible or broken basal membrane. 3. sham operation group and ischemia-reperfusion group for different time periods (0/ 1/ 2/ 4h) serum C There was no significant difference in NP content (n = 8, P0.05). The expression of NPR-B was highest (n = 8, P 0.05). 5. Both the sham operation group and the renal tissue NPR-B of the ischemia-reperfusion group were positive. Compared with the reperfusion group, the positive brown granules increased significantly, and the staining was significantly increased. The expression of NPR-B mRNA in renal tissues of rats was significantly higher than that in normal control group. The dirty volume is obviously increased and the quality is soft; the section is seen in the medulla of the kidney; the renal medulla is dark red; the cortex is swollen and pale. The near-end tubular epithelial cell vacuoles and particle degeneration under the microscope, the cell is flat, the tube cavity is expanded, the brush-like edge falls off, and the exfoliated epithelium, the tube and the tube cavity are formed in the tube cavity. Inflammatory exudate, bare basement membrane, and even segmental focal necrosis of tubular cells. Renal tubular stroma can have varying degrees of inflammatory cells. Infiltration and edema, visible cell debris or particle casts in the distal tubules and manifold lumen. Tubular basement membrane segment fracture, fracture Site visible lymphocytes, monocytes, and neutralizing Neutrophil infiltration, renal interstitial diffuse edema. No significant changes in glomerular and renal function were found. Renal tubular arrangement of CNP group After 24 hours of ischemia-reperfusion group, the level of serum Cr and NPY was significantly higher than that of sham-operated group (n = 8, P0.001). (n = 8, P0.05). Compared with the sham operation group, the content of MDA increased significantly and the activity of SOD decreased significantly (n = 8, P0.01 and P0.05). Compared with sham operation group and CNP, the expression of Bax was significantly increased (n = 8, P0.05). Significant differences, but C The ratio of Bax/ Bcl-2 decreased (n = 8, P0.05). Results: 1. The structure and function of tissue were obviously damaged during renal ischemia-reperfusion. especially at the junctional region and medulla of the skin. The expression of NPR-B was obviously up-regulated. To improve the structure and function of kidney tissue damaged by ischemia-reperfusion injury, and present an anti-injury effect.
【学位授予单位】：延边大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
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