【作者】 宋涛；

【导师】 葛志明；

【作者基本信息】 山东大学 ， 内科学（心血管病）（专业学位）， 2019， 博士

【摘要】 研究背景据2017年《中国心血管病报告》推算,目前我国心血管病患者人数已达2.9亿,心血管疾病死亡人数占居民死亡人数的40%以上,其死亡率已高居首位,高于肿瘤和其他疾病。随着人们生活方式的改变和生活水平的提高,冠心病已经成为严重危害人类健康的重大疾病。由于心血管疾病的发生率逐年升高,而且心力衰竭(heart failure,HF)是许多冠心病患者的最终转归,因此心力衰竭已成为21世纪心血管医生所面临的重大挑战。随着冠脉介入技术的迅速发展以及处理复杂病变能力的不断提高,越来越多的心衰患者通过经皮冠状动脉介入治疗(per cutaneous coronary intervention,PCI)技术减少心肌细胞的缺血缺氧,从而明显改善患者心功能和预后。随着对比剂的应用日趋广泛以及用量不断增加,对比剂肾病(contrast-induced nephropathy,CIN)的发生率也随之明显增加。CIN的临床特征表现为血清肌酐水平在应用对比剂后24小时内逐渐升高,2～3天达到峰值,7～10天内血清肌酐下降至基线水平。临床上CIN通常定义为在排除其他肾损害因素的情况下,患者应用对比剂后2～3d内出现急性肾功能损害性疾病,具体的诊断标准为应用对比剂后2～3d内血清肌酐较基础值升高>25%或升高44.2umol/L(0.5mg/dl)。对比剂肾病已成为医院获得性急性肾功能不全的第三大常见原因,仅次于肾灌注不足和肾毒性药物引起的肾损害,是临床上应用含碘对比剂后引起的最严重并发症之一。CIN患者不仅并发症多、住院时间长,而且预后差、死亡率高。既往研究表明普通患者应用对比剂后,CIN总的发病率大约2～3%。但在高龄、糖尿病、慢性肾病、慢性心力衰竭、低血压及应用肾毒性药物等高危患者,CIN的发病率高达11%～50%。由于目前CIN尚无特异性治疗措施,因此早期进行危险分层、识别高危患者,积极采取预防措施显得尤为重要。目前预防C IN的可能有效措施包括:首选应用等渗对比剂,尽量减少对比剂用量,充分给予水化治疗,避免应用肾毒性药物,给予他汀预处理,积极应用抗氧化剂如N-乙酰半胱酸、还原型谷胱甘肽、维生素C等。目前CIN的诊断标准主要依据血清肌酐(SCr)的升高水平,但由于SCr受多种因素的影响,其敏感性差,不能够早期诊断CIN,从而延缓CIN的早期治疗,因此临床上需要应用一些能够早期反应肾损害的生物学标志物来代替SCr。目前临床上能够反应CIN早期肾损害的标志物有:胱抑素C(CysC)、肾损伤分子-1(KIM 1)、白细胞介素-18(IL-18)、β2-微球蛋白(β2-MG)、中性粒细胞明胶酶相关脂质运载蛋白(NGAL)、N-乙酰-β-D-葡萄糖苷酶(NAG)、肝脏型脂肪酸结合蛋(L-FABP)等。研究表明,这些早期生物学标志物有助于早期发现、早期诊断、早期治疗对比剂肾病,具有较好的敏感性及特异性。本研究不仅检测尿素氮(BUN)、肌酐(Scr),估算肾小球滤过率(eGFR)等传统反应肾功能损害的指标,而且还联合检测能够早期反映肾损害的血清CysC、尿NAG酶和尿β 2-微球蛋白三个新型标志物,通过收集相关肾功能、肾小管功能变化指标进行对比分析,比较两组患者之间肾损害的严重程度和对比剂肾病的发生率。既往研究表明患者应用高渗对比剂后CIN的发生率较高,而应用低渗或等渗对比剂后CIN的发生率明显低于高渗对比剂,因此临床上更倾向选用低渗或等渗对比剂。目前究竟是低渗对比剂还是等渗对比剂诱发CIN的发生率低,近年来的meta分析结论并不一致。有的研究表明应用等渗对比剂较低渗对比剂具有更低的CIN发生率,有的研究表明应用等渗对比剂与低渗对比剂诱发CIN的发生率无明显差异。在目前临床应用的对比剂中,大多数的观点认为等渗对比剂碘克沙醇的肾毒性最低。美国ACC/AHA制定的临床指南,在慢性肾病合并急性冠脉综合征的患者中将碘克沙醇列为I类推荐、A级证据。一些随机对照试验表明,在高龄、糖尿病、慢性肾病患者中行冠脉介入治疗时,等渗对比剂较低渗对比剂具有更低的CIN发生率。但在冠心病合并慢性心力衰竭患者中,目前应用等渗对比剂是否同样能够降低CIN的发生率还不清楚。本研究目的是在冠心病合并慢性心力衰竭患者行冠脉介入治疗时,对比应用等渗对比剂与低渗对比剂诱发肾损害的严重程度CIN的发生率,而且还联合检测早期反映肾损害的血清CysC、尿NAG酶和尿β2-微球蛋白三个新型标记物来评价肾损害的严重程度,从而为冠心病合并慢性心力衰竭患者更好地预防CIN的发生,也为这类患者安全进行冠脉介入治疗提供理论依据。目前CIN的发病机制尚未明确,认为可能是肾髓质缺血缺氧损伤、对比剂对肾小管细胞的直接毒性作用、氧化应激以及炎性反应等多因素共同参与的结果。本课题还应用对比剂处理人肾近端小管上皮细胞,分析细胞增殖、凋亡变化,比较细胞炎症、凋亡相关基因和蛋白表达,分析不同类型对比剂引起肾细胞损伤的可能机制。同时还构建小鼠急性肾损伤模型,通过尾静脉注射等渗或低渗对比剂,体内分析两种对比剂对于肾功能的影响和可能分子机制。研究目的1.在冠心病合并慢性心力衰竭患者行冠脉介入治疗时,比较应用等渗与低渗对比剂出现肾损害的严重程度和CIN的发生率。2.联合检测早期反映肾损害的血清CysC、尿NAG酶和尿β 2-微球蛋白三个新型标记物来评价对比剂肾病患者的肾损害程度。3.通过细胞实验和动物模型研究碘克沙醇和碘海醇诱发肾损害的分子机制,进一步阐明应用等渗与低渗对比剂时肾损害程度差异的原因。资料与方法1.研究对象:选取2015年1月～2016年5月在滕州市中心人民医院心内科收治的220例冠心病合并慢性心力衰竭患者,入选患者行冠状动脉造影(CAG)或经皮冠状动脉介入治疗(PCI),其中男性138例,女性92例,年龄在35～77岁之间,平均年龄(53.51±9.37)岁。所有入组患者均处于心力衰竭的稳定期且LVEF在30%～50%之间,按照《中国心力衰竭诊断和治疗指南2014》中慢性心力衰竭诊断标准。剔除标准:怀孕或哺乳期妇女、有对比剂过敏史、急性心肌梗死、急性左心衰、血流动力学不稳定、近5d内应用过对比剂、糖尿病患者术前3d内服用过二甲双胍、严重肝功能不全、行透析治疗的终末期肾病患者、恶性肿瘤患者等。本研究已通过滕州市中心人民医院伦理委员会审查批准,所有入组患者术前均签署知情同意书。人肾近端小管细胞系HK-2细胞购买自中国科学院上海生命科学研究院细胞库。6-8周龄的雄性C57BL/6N小鼠购买自上海Slac实验动物中心。2.研究对象分组:本研究为前瞻性、单盲、随机研究,将入选220例患者按随机数字表法分为两组:对照组(1 10例)和观察组(1 10例)。对照组应用江苏扬子江药业公司生产的低渗对比剂碘海醇(iohexol),男性57例,女性53例,年龄在32～75岁之间,平均年龄(52.36±10.60)岁。观察组应用江苏恒瑞医药公司生产的等渗对比剂碘克沙醇(iodixanol),男性61例,女性49例,年龄在38～79岁之间,平均年龄(54.16±9.82)岁。6-8周龄的雄性C57BL/6N小鼠并饲养于无特定病原体的条件下,随机分配到实验动物组。3.研究方法3.1临床资料收集:收集并记录入组患者基础临床资料:性别、年龄、身高、体重、体重指数、血压数值、既往有无高血压或糖尿病史、心功能指标、肾功能指标、治疗心衰所用药物、应用对比剂剂量、静脉水化剂量等。3.2主要观察指标:在术前所有患者均常规行心脏彩超、N-末端B型钠尿肽(NT-proBNP)检查评价心功能。术前常规化验三大常规(血液、尿液、大便)、血生化、CysC、尿NAG酶、β2-MG及高敏C反应蛋白(hsCRP)。术后第1天、第3天复查尿常规、肾功能、NT-proBNP、CysC、尿NAG酶及β-MG及hsCRP。心内科医师按规范行CAG术,并按标准方法行PCI术。所有入选患者在心功能耐受的情况下至少术前及术后6小时内给予0.9%生理盐水以0.5ml/(kg·h)速度常规静脉滴注,同时鼓励患者尽量多饮水。依据分组不同分别选用不同的对比剂,对照组和观察组分别应用碘海醇和碘克沙醇,并记录对比剂的应用剂量。主要观察终点是两组患者术后CIN的发生率。次要观察终点是两组患者术后第1天、第3天BUN、SCr、估计肾小球滤过率(eGFR)、CysC、尿NAG酶、β2-MG、尿比重及hsCRP变化水平。估计肾小球滤过率(estimated glomerular filtration rate,eGFR)计算公式采用简化MDRD公式:估计肾小球滤过率[ml/(min·1.73m2)]=186×(血清肌酐)-1.154×(年龄)-0.203 ×(0.742女性)。血清Cr单位为mg/dl,年龄单位为岁。3.3细胞实验:以MTT法检测细胞增殖,TUNEL法检测细胞凋亡,定量逆转录-聚合酶链式反应(RT-PCR)检测炎症、凋亡相关mRNA的表达,Western blot实验检测炎症、凋亡相关蛋白的表达。3.4急性肾损伤小鼠模型构建:将C57BL/6雄性小鼠随机分成六组:(1)A组,对照组(n=6);(2)B组,单侧肾切除术模型(n=6);(3)C组,单侧肾切除模型+断水1天+0.9%生理盐水(10μL/g,n=6);(4)D组,单侧肾切除模型+断水1天+呋塞米注射液(10 μL/g,n=6);(5)E组,单侧肾切除模型+断水1天+呋塞米注射液(10 μL/g)+碘海醇(10μL/g,n=6);(6)F组,单侧肾切除模型+断水1天+呋塞米注射液(10μL/g)+碘克沙醇(10μL/g,n=6)。模型构建完成3周后进行尾静脉注射对比剂,于对比剂注射前、注射后24h收集小鼠血液,检测小鼠肾功能指标(Scr、BUN、Ccr)。对比剂注射后24h处死小鼠,收集小鼠肾组织,用HE染色、RT-PCR、Western blot方法对肾组织进行后续检测。4.统计学方法:将本研究中所有实验数据输入EXECL表格,并使用SPSS 20.0软件进行统计分析。计量资料以x+s表示,两样本间比较采用Student’s t-test检验,多组间比较采用ANOVA检验。计数资料以百分率(%)表示,组间比较采用Chi-square test检验,P<0.05为差异有统计学意义。结果1.对比剂对于冠心病慢性心力衰竭患者肾功能的影响1.1两组患者的基础临床资料、心功能指标、肾功能指标、治疗心衰所用药物、对比剂用量、静脉水化用量均无统计学差异(P>0.05),两组之间具有可比性。1.2两组患者肾功能指标的比较 低渗组与等渗组之间的血清BUN、Scr、eGFR、CysC术前无统计学差异(P>0.05)。低渗组与等渗组之间血清BUN、Scr在术后第1天均有升高,但无明显统计学差异,而低渗组与等渗组之间eGFR、CysC在术后第1天具有统计学差异(P<0.05)。低渗组与等渗组之间的血清BUN、Scr、eGFR、CysC在术后第3天均具有统计学差异(P<0.05)。1.3两组患者肾小管功能的比较 低渗组与等渗组之间尿NAG酶、β2-MG、尿比重在术前无统计学差异(P>0.05)。低渗组与等渗组之间尿NAG酶、β2-MG在术后第1天、第3天均有升高,且两组间尿NAG酶、β2-MG差异具有统计学意义(P<0.05)。低渗组与等渗组之间尿比重在术后第1天、第3天较前逐渐降低,但无统计学差异(P>0.05)。1.4两组患者高敏C反应蛋白的比较 低渗组与等渗组之间hsCRP在术前无统计学差异(P>0.05)。低渗组与等渗组之间hsCRP在术后第1天、第3天均明显升高,两组间具有统计学差异(P<0.05)。1.5两组患者对比剂肾病发生率的比较低渗组和等渗组两组患者总共发生46例CIN,CIN总的发生率为20.9%。其中低渗组患者发生32例CIN,CIN发生率为29.1%,等渗组患者发生14例CIN,CIN发生率为12.7%,两组间CIN的发生率具有明显统计学差异(P<0.05)。在亚组分析中,低渗组行PCI患者CIN的发生率为36.2%,等渗组行PCI患者CIN的发生率为16.3%,两组间CIN的发生率具有明显统计学差异(P<0.05)。低渗组未行PCI患者CIN的发生率为23.8%,等渗组未行PCI患者CIN的发生率为9.8%,两组间CIN的发生率具有明显统计学差异(P<0.05)。在PCI和未行PCI患者的亚组分析中,等渗组患者CIN的发生率明显低于低渗组患者,两组间CIN的发生率具有明显统计学差异(P<0.05)。2.对比剂对于人肾近端小管细胞的影响碘海醇/碘克沙醇处理细胞后,HK-2细胞增殖较OmgI/mL对比剂处理的对照组明显降低(P<0.05),在40～120mgI/mL浓度区间内,碘海醇处理的HK-2细胞增殖较碘克沙醇处理组明显减少(P<0.05);含有120mgI/mL的碘海醇/碘克沙醇,处理细胞1h、2h、4h、6h、8h、10h、12h后发现,HK-2细胞增殖随处理时间延长明显降低(P<0.05)。与对照组相比,碘海醇与碘克沙醇组细胞凋亡率均明显增加,其中碘克沙醇组细胞凋亡率较碘海醇组明显降低(P<0.05)。碘海醇/碘克沙醇处理后,炎症相关ED-1、TNF-ααmRNA表达明显增加,碘克沙醇组ED-1、TNF-α mRNA表达较碘海醇组明显降低(P<0.05)。凋亡相关Bax、caspase-3 mRNA表达明显增加,抗凋亡相关Bcl-2mRNA表达明显降低,碘海醇组变化趋势较碘克沙醇组更加明显(P<0.05)。碘海醇/碘克沙醇处理组与对照组相比,炎症相关蛋白ED-1、TNF-α表达明显增加,凋亡相关蛋白Bax、cleaved-caspase-3表达明显增加,抗凋亡相关蛋白Bcl-2表达明显降低,且碘海醇处理组较碘克沙醇处理组各蛋白变化趋势更加明显(P<0.05)。3.对比剂对于小鼠肾功能、肾组织及肾组织基因蛋白表达的影响B～F组小鼠对比剂注射前肾功能指标Scr、BUN明显高于对照组A组,而Ccr则明显低于对照组(P<0.05),B～F组间各指标未见明显差异(P>0.05)。各组按预定方案处理后24h,B～D组小鼠肾功能指标较处理前无明显差异(P>0.05),E、F组小鼠肾功能指标Scr、BUN较注射前明显升高、Ccr较对照组明显降低,其中E组小鼠肾功能指标较F组变化更明显(P<0.05)。在A～F组小鼠肾组织HE染色后发现细胞损伤逐渐加重,E、F组小鼠肾组织细胞损伤均较D组更为明显,其中以E组细胞损伤最为严重。与对照组A组相比,B～F组小鼠肾组织炎症相关ED-1、TNF-ααmRNA表达均明显增加(P<0.05),B～D组间炎症相关ED-1、TNF-ααmRNA表达无统计学差异(P>0.05),E、F组炎症相关ED-1、TNF-ααmRNA表达均较D组明显增加,而E组ED-1、TNF-α表达较F组明显增加,差异均有统计学意义(P<0.05)。与对照组A组相比,B～F组小鼠肾组织凋亡相关Bax、caspase-3 mRNA表达均明显增加,抗凋亡相关Bcl-2mRNA表达均明显降低,差异有统计学意义(P<0.05)。B～D组凋亡相关Bax、caspase-3 mRNA及抗凋亡相关Bcl-2mRNA表达无统计学差异(P>0.05)。与D组相比,E、F组凋亡相关Bax、caspase-3mRNA表达明显增加,抗凋亡相关Bcl-2mRNA表达明显降低,差异有统计学意义(P<0.05),E组Bax、caspase-3mRNA表达明显高于F组,Bcl-2mRNA表达则明显低于F组,差异均有统计学意义(P<0.05)。与对照组A组相比,B～F组炎症相关蛋白ED-1、TNF-α表达明显增加,凋亡相关蛋白Bax、cleaved-caspase-3表达明显增加,抗凋亡相关蛋白Bcl-2表达明显降低(P<0.05),B～D组间各蛋白表达无统计学差异(P>0.05)。E、F组与D组相比,炎症相关蛋白ED-1、TNF-α表达明显增加,凋亡相关蛋白Bax、cleaved-caspase-3表达明显增加,抗凋亡相关蛋白Bcl-2表达明显降低,且E组较F组各蛋白变化趋势更明显(P<0.05)。结论1.在冠心病慢性心力衰竭患者进行冠脉介入治疗时,等渗对比剂较低渗对比剂能够明显降低CIN的发生率,因此等渗对比剂具有更低的肾毒性。联合检测早期反映肾损害的血清CysC、尿NAG酶和尿β2-MG三个新型标记物能够全面、精确地评价肾损害的严重程度。2.对比剂可抑制人肾近端小管细胞增殖,增加了肾小管细胞的炎症反应和细胞凋亡,低渗性碘海醇较等渗性碘克沙醇对细胞增殖、炎症、凋亡的影响更显著。3.对比剂可造成小鼠肾组织细胞损害,增加了小鼠肾组织的炎症反应和细胞凋亡,低渗性碘海醇较等渗性碘克沙醇对小鼠肾组织细胞损害更显著。更多还原

【Abstract】 Background:Nowadays the population with cardiovascular disease has reached 290 million,according to 2017《China cardiovascular disease report》.Cardiovascular death account for more than 40%of the death of residents’ disease.The cardiovascular mortality rate ranks first,higher than cancer and other diseases.With the changes of people’s lifestyles and the improvement of living standards,coronary heart disease has becomed a main disease seriously harmful to human health.Because the incidence of cardiovascular disease increased year by year,heart failure is the final outcome of many patients with coronary heart disease,which has become a major challenge for cardiovascular physicians in the 21st century.With the rapid development of percutaneous coronary intervention and the ability to treat complex coronary lesions,more and more heart failure patients have reduced myocardial ischemia and hypoxia through the percutaneous coronary intervention(PCI)technology,which can signify cantly improve cardiac function and prognosis.As the use of contrast agent becomes more widespread and the dosage of contrast agent continues to increase,the incidence of contrast-induced nephropathy(CIN)is increasing consequently.The typical clinical feature of CIN is that serum creatinine increased gradually within 24 hours after the application of contrast agent,reached a high peak in 2～3 days,and serum creatinine decreased to baseline level within 7～10 days.CIN is usually defined as the occurrence of acute renal dysfunction in patients within 2～3 days after the application of contrast agent in the absence of other factors of renal damage.The specific diagnostic criteria is a relative increase>25%from-baseline of serum creatinine or an absolute increase>44.2μmol/L(0.5mg/dL)in serum creatinine within 2～3 days after application of contrast agent.Contrast-induced nephropathy has become the third most common cause of hospital-acquired acute renal insufficiency,second only to renal perfusion and nephrotoxic drugs,which is the most serious complication caused by the clinical application of iodine-containing contrast agents.For patients who develop CIN following PCI,the prognostic impact is substantial.In addition to renal complications,more complications,prolonged hospital stays,poor prognosis and higher in-hospital mortality rates have been reported among patients who develop CIN as compared with those who don’t.Previous studies have shown that the incidence of CIN is approximately 2～3%for the general patients who undergoing coronary procedures.However,the incidence of CIN can reach from 11%to 50%in high-risk patients such as advanced age,diabetes,chronic kidney disease,heart failure,hypotension,and the application of nephrotoxic drugs.Since there is no specific treatment for CIN at present,it is particularly important to correctly understand the risk factors of CIN,identify high-risk patients and give appropriate preventive measures in the early stage.At present,the possible effective measures to prevent CIN include as follows:the preferred application of isotonic contrast agent,minimize the amount of contrast agent,adequately give hydration treatment,avoid the use of nephrotoxic drugs,apply statins pretreatment and active application of antioxidant drugs such as N-acetylcysteic acid、reduced glutathione、vitaminsC and so on.At present,the diagnostic criteria of CIN are mainly based on the elevated level of serum creatinine(SCr).But because of its susceptible to many factors and poor sensitivity,it is not possible to diagnose CIN early,thus delaying the early treatment of CIN.Therefore,some biological markers of early renal damage are needed clinically to replace SCr.Currently,there are clinical markers that can reflect early renal damage in CIN as follows:CystatinC,Kidney injury molecule-1(KIM-1),Interleukin-18(IL-18),β2-Microglobulin(β2-MG),Neutrophil gelatinase-asssociated lipocalin(NGAL),N-acety-beta-D-glucosaminidase(NAG enzyme),Liver-type fatty acid binding protein(L-FABP)and so on.Some clinical studies have shown that these early biomarkers is heipful for early detection、early diagnosis and early treatment of CIN,which have better sensitivity and specificity.Our study not only detected the traditional indexes of renal dysfunction such as urea nitrogen(BUN)、creatinine(Scr)、estimated glomerular filtration rate(eGFR),but also combined detection three new early-stage renal dysfunction indexes of serum cystatin C,urinary NAG enzyme and urinary β2-microglobulin to early comprehensively evaluate the renal damage in CIN patients.This study compared the severity of renal damage and the incidence of of CIN between the two groups by collecting relevant functional indicators of renal function and renal tubular function for observation and analysis.Previous clinical studies have shown that high-osmolar contrast agent have a higher incidence of CIN.The incidence of CIN induced by low-osmolar contrast agent and iso-osmolar contrast agent is significantly lower than that of the high-osmolar contrast agent,Therefore,it is more clinically preferred to choose low-osmolar contrast agent or iso-osmolar contrast agent.At present,whether the incidence of CIN induced by low-osmolar contrast agents is low or the incidence of CIN induced by iso-osmolar contrast agents is low,meta-analysis has not reached a consistent conclusion recently.Some studies have found that the iso-osmolar contrast agents have lower in the incidence of CIN,and some studies have shown that the iso-osmolar contrast agents have no significant difference in the incidence of CIN with low-osmolar contrast agent.Although the results of some clinical trials are inconsistent,most of the viewpoints are that that nonionic iso-osmolar contrast agent iodixanol has lowest nephrotoxicity mong the currently used contrast agents for clinical use.A clinical guideline developed by the American Heart Association/American Heart Association classifies iodixanol as a Class I recommendation,Class A evidence in patients with chronic kidney disease and acute coronary syndrome.Some randomized controlled trials have shown that iso-osmolar contrast agent have lower nephrotoxicity in coronary interventions in elderly,diabetic,and chronic kidney disease patients.However,it is unclear whether the use of iso-osmolar contrast agent can reduce the incidence of contrast nephropathy in patients with coronary heart disease and chronic heart failure.This study aims to compare the severity of renal damage and the incidence of of CIN in patients after administration of the iso-osmolar iodixanol or low-osmolar iohexol in patients with coronary heart disease andchronic heart failure undergoing coronary intervention.At the same time,the combination of detecting serum cystatinC,urinary NAG enzyme and urinary β2-microglobulin is used to early assess the severity of renal damage.We hope this study is to better prevent the occurrence of CIN in patients with coronary heart disease and chronic heart failure,and also provide a theoretical basis for safe coronary intervention for such patients.At present,the pathogenesis of CIN is still unclear,and it may be the result of multiple factors including renal medullary ischemia and hypoxia injury,direct toxicity of contrast agenton renal tubular cells,oxidative stress and inflammatory response.This study also used contrast agent to treat human renal proximal tubular epithelial cells,analyze cell proliferation and apoptosis,compare cell inflammation,apoptosis-related genes and protein expression,and analyze the possible mechanisms of renal cell damage caused by different types of contrast agents.At the same time,a model of acute kidney injury in mice was constructed.The effects of two contrast agents on renal function and possible molecular mechanisms were analyzed in vivo by injection of iso-osmolar contrast agent or low-osmolar contrast agent in the tail vein.Objective1.To compare the severity of renal damage and the incidence of of CIN after administration of the iso-osmolar iodixanol or low-osmolar iohexol in a population of patients with coronary heart disease and chronic heart failureundergoing coronary intervention procedure.2.Combined detection three new markers of serum cystatin C,urinary NAG enzyme and urinary β2-microglobulin to early evaluate the severity of renal damage in patients with contrast nephropathy.3.The molecular mechanism of renal damage induced by iodixanol and iohexol was studied by cell experiments and animal models,and the reasons for the different extent of renal damage between iso-osmolar and low-osmolar contrast agent were further elucidated.Materials and Methods1.Study populationsThis clinical trial was conducted at the department of cardiology in Tengzhou central people’s hospital in Shandong Province of China from January 2015 to May 2016.Patients referred for coronary angiography or PCI were considered to be eligible if they had a history of coronary heart disease and chronic heart failure.They included two hundred and twenty heart failure patient,including 138 men,92 women,age rang 35～77 years,mean age 53.51±9.37 years.All enrolled patients were in stable phase of heart failure with LVEF ranged from 30%to 50%,according to the diagnostic criteria for chronic heart failure in the the 2014 edition of《the Chinese Heart Failure Diagnosis and Treatment Guidelines》.Exclusion criteria were pregnancy or lactation,allergy to contrast agent allergy,acute myocardial infarction,acute heart failure,hemodynamic instability,acute left ventricular failure,five days applied contrast agent,three days before taking metformin in diabetes patients,severe liver dysfunction,end stage renal disease dialysis treatment of patients,malignant tumor patients and so on.The study protocol was reviewed and approved by the local ethics committee.All enrolled patients signed informed consent before the procedure.The human renal proximal tubule cell line HK-2 cells were purchased from the stem cell bank of the Shanghai Institute of Biological Sciences.The male C57BL/6N mice 6～8 weeks old were purchased from the Shanghai Slac Experimental Animal Center.2.Study protocolThis study is a prospective,single-blind,randomized study.The selected two hundred and twenty consecutive patients were randomly assigned to the control group and the observation group with sealed envelopes than contained a computer generated randomization sequence,with 110 patients in each group.The control group used the low-osmolar contrast agent iohexol produced by Jiangsu Yangzijiang Pharmaceutical Co.Ltd,which includs 57 males and 53 females,age rang 32～75 years,mean age(52.36±10.60)years.The observation group applied the iso-osmolar contrast agent iodixanol produced by Jiangsu Hengrui Pharmaceutical Co.Ltd.,which includs 61 males and 49 females,aged rang 38～79 years,mean age(54.1 6±9.82)years.The male C57BL/6N mice 6-8 weeks old were purchased from the Shanghai Slac Experimental Animal Center,which were randomly assigned to experimental animals under conditions free of specific pathogens.3.Methods3.1 Clinical data collectionAll patients were asked about medical history in detail,recorded the gender,age,height,weight,blood pressure,previous history of hypertension or diabetes,cardiac function index,renal function index,drugs for treating heart failure,the volumes of contrast agent and the volumes of intravenous hydration.3.2 Main observation indicatorsAll selected patients underwent routine cardiac ultrasonography and N-terminal B-type natriuretic peptide(NT-proBNP)examination to evaluate cardiac function before procedure.All patients were tested the parameters as follows:three routine tests(blood、urine and stool),blood biochemistry,CysC,N-acety-beta-D-glucosemine-dase(NAG),β2-Microglobulin and high sensitivity C-reactive protein(hsCRP)before routine proedure.The urine routine,renal function,NT-proBNP,CysC,urinary NAG enzyme,β2-MG and hsCRP were detected on the first and third day after the procedure.Coronary angiography or angioplasty were performed according to standard protocols for our institution using the radial approach.All enrolled patients were given regular intravenous infusion of 0.9%saline at a rate of 0.5 ml/(kg·h)at least 6 hours before and 6 hours after procedure,and patients were encouraged to drink as much water as possible.The iso-osmolar contrast agent iodixanol and low-osmolar contrast agent iohexol were selected according to the divided group,and the amount of the contrast agent was recorded.The primary endpoint of this study was the incidence of CIN between the two groups after procedure.BUN,SCr,estimated glomerular filtration rate(eGFR),CysC,urinary NAG enzyme,β2-MG,urine specific gravity,and hsCRP.The secondary study endpoints were peak increase in the level of BUN,SCr,CysC,urinary NAG enzyme,β2-MG,urine specific gravity,hsCRP and the decrease in the level of estimated glomerular filtration rate(eGFR)on the first and third day after the procedure.Estimated glomerular filtration rate was calculated using the simplified MDRD formula:Estimated Glomerular filtration rate(eGFR)[ml/(min·1.73m2)]=186x(serumcreatinine)-1.154×(age)-0.203×(0.742female).the unit of serum creatinine is mg/dl,the unit of age is year.3.3 Cell experimentsCell proliferation was detected by MTT assay,apoptosis was detected by TUNEL assay,and expression of inflammation and apoptosis related mRNA was detected by RT-PCR.Expression of inflammation and apoptosis related proteins were detected by Western blot.3.4 Construction of a mouse model of acute kidney injuryC57BL/6 male mice were randomly divided into six groups:(1)group A,control group(n=6);(2)group B,unilateral nephrectomy model(n=6);(3)Group C,unilateral nephrectomy model+water cut lday+0.9%saline(10μL/g,n=6);(4)group D,unilateral nephrectomy model+water cut 1day+Furosemide injection(10μL/g,n=6);(5)Group E,unilateral nephrectomy model + water cut 1 day + furosemide injection(10μL/g)+iohexol(10μL/g,n=6);(5)Group F,unilateral nephrectomy model + water cut 1 day+furosemide injection(10μL/g)+iodixanol(10μL/g,n=6).Three weeks after the model was constructed,the contrast agent was injected into the tail vein.The urine of the mice was collected before the contrast injection and 24 hours after the injection.The renal function indexes(Scr,BUN,Ccr)were detected,and the mice were sacrificed 24 hours after the contrast injection.The mouse kidney tissue was collected.The kidney tissue was subjected to subsequent detection by HE staining,,RT-PCR and Western blot.4.Statistical analysisAll experimental data in this study were entered in the EXECL form,which were statistically analyzed by using SPSS 20.0 software Program.Continuous variables were described as mean±standard deviation.The Student’s t-test is used for comparison between the two samples.Comparison among multi-groups were performed with ANOVA test.Categorical variables was described as percentage.Categorical difference was analyzed by Chi-squared test.P<0.05 was considered statistically significant.Results1.The effects of contrast agent on renal function in patients with coronary heart disease and chronic heart failure1.1 Patient demographics and dispositionThere was no significant difference in basic clinical data,cardiac function indicators,renal function indicators,drugs for treating heart failure,the volumes of contrast agent and the volumes of intravenous hydration between the two groups before procedure(P>0.05).The two groups were comparable.1.2 Comparison of renal function indexes between the two groupsThere was no significant difference in BUN,SCr,eGFR and CysC between the low-osmolar group and the iso-osmolar group before procedure(P>0.05).There was no significant difference in BUN and SCr,but there was statistically significant difference in eGFR and CysC between the two groups on the first day after procedure(P<0.05).There was statistically significant difference in BUN,SCr,eGFR and CysC between the two groups on the third day after procedure(P<0.05).1.3 Comparison of renal tubular function indexes between the two groupsThere was no significant difference in urine NAG,P2-MG and SG between the low-osmolar group and the iso-osmolar group(P>0.05).The levels of NAG and P2-MG in the two groups significantly increased,and there was significant difference on the first and third day(P<0.05).The SG of urine in the two groups was downtrend on the first and third day,but there was no significant difference(P>0.05).1.4 Comparison of high sensitive C-reactive protein between the two groupsThere was no significant difference in high sensitive C-reactive protein between the low-osmolar group and the iso-osmolar group before procedure(P>0.05).There was significant difference in hsCRP between the first day and the third day after procedure(P<0.05).1.5 Incidence of contrast-induced nephropathy between the two groupsThere were 46 cases of contrast-induced nephropathy in the two groups.The overall incidence of CIN was 20.9%.Among them,32 patients with CIN occurred in the low-osmolar group,the incidence of CIN was 29.1%,14 patients with CIN in the iso-osmolar group,and the incidence of CIN was 12.7%(P<0.05).In the subgroup analysis of patients with PCI,the incidence of CIN in the low-osmolar group was 36.2%,and the incidence of CIN in the iso-osmolar group was 16.3%(P<0.05).In the subgroup analysis of patients without PCI,the incidence of CIN in the low-osmolar group was 23.8%,and the incidence of CIN in the iso-osmolar group was 9.8%(P<0.05).In the subgroup analysis of patients with PCI or non-PCI,the incidence of CIN in the iodixanol group was significantly lower than that of the iohexol group,and the difference of the incidence of CIN between the two groups was statistically significant(P<0.05).2.The effects of contrast agent on human renal proximal tubule cellHK-2 cell proliferation was significantly lower than that of OmgI/mL contrast agent(P<0.05).In the concentration range of 40～120mgI/mL,the proliferation of HK-2 cells treated with iohexol was significantly lower than that of the iodixanol treatment group(P<0.05).After treatment of cells for 1h、2h、4h、6h、8h、10h、12h containing 120mgI/mL of iohexol/ioxolol,it was found that the proliferation of HK-2 cells decreased significantly with the treatment time(P<0.05).Compared with the control group,the apoptosis rate of iohexol and iodixanol groups increased significantly,and the apoptosis rate of iodoxanol group was significantly lower compared with the iohexol group(P<0.05).After treatment with iohexol/ioxolol,the expressions of inflammation-related ED-1 and TNF-α mRNA were significantly increased.The expressions of inflammation-related ED-1 and TNF-α mRNA in the iodixanol group were significantly lower than that of the iohexol group(P<0.05).The expressions of apoptosis-related Bax and caspase-3 mRNA were significantly increased,and the expression of anti-apoptosis-related Bcl-2 mRNA was significantly decreased(P<0.05),and the change trend was more obvious than that of iodixanol(P<0.05).Compared with the control group,the expressions of inflammation-related proteins ED-1 and TNF-α were significantly increased in the iohexol/ioxolol treatment group,and the expressions of apoptosis-related proteins Bax and cleaved-caspase-3 were significantly increased.The expression of anti-apoptosis-related protein Bcl-2 was significantly decreased,and the trend of each protein in the iohexolol treatment group was more obvious than that in the iodixanol treatment group(P<0.05).3.The effects of contrast agent on renal function,renal tissue and expression of renal tissue gene protein in miceCompared with the control group A,the level of Scr and BUN in the B～F group was significantly higher,while the Ccr was significantly lower(P<0.05).There was no significant difference in the level of Scr、BUN and Ccr between the B～F groups(P>0.05).There was no significant difference in the renal function indexes of the B～D group before treatment(P>0.05).The renal function indexes Scr and BUN of the E and F group were significantly higher than those before injection,and the Ccr was significantly higher than that of the control group A.The renal function of group E was significantly higher than that of group F(P<0.05).In each group of mice,the group E and F mice showed obvious cell damage after HE staining,which was more obvious than that of group D mice.Compared with the control group A,the expressions of inflammation-related ED-1 and TNF-a mRNA in the kidney tissues of the B～F groups were significantly increased(P<0.05).There were no significant differences in ED-1 and TNF-a mRNA among the B、C and D groups(P>0.05).The expressions of inflammation-related ED-1 and TNF-α mRNA in the kidney tissues of the group E and F were significantly higher compared with the group D(P<0.05).The expressions of inflammation-related ED-1 and TNF-amRNA in kidney tissues of group E were significantly higher than that of group F(P<0.05).Compared with the control group A,the expressions of apoptosis-related Bax and caspase-3 mRNA in renal tissues of mice in B-F groups were significantly increased,and the expression of anti-apoptosis-related Bcl-2 mRNA was significantly decreased(P<0.05).There were no significant differences in Bax、caspase-3 and Bcl-2 mRNA among the B、C and D groups(P>0.05).Compared with the control group D,the expressions of apoptosis-related Bax and caspase-3 mRNA in renal tissues of mice in E and F groups were significantly increased,and the expression of anti-apoptosis-related Bcl-2 mRNA was significantly decreased(P<0.05).The expression of apoptosis-related Bax and cleaved-caspase-3 of group E was significantly higher than that of group F,and the expression of Bcl-2 was significantly lower than that of group F(P<0.05).Compared with the control group A,the expressions of inflammation-related proteins ED-1 and TNF-α in the B～F groups were significantly increased(P<0.05).The expressions of apoptosis-related proteins Bax and cleaved-caspase-3were significantly increased,and the expression of anti-apoptosis-related protein Bcl-2 was significantly decreased(P<0.05).There were no significant differences in protein indexes among the B、C and D groups(P>0.05).Compared with group D,the expressions of inflammation-related proteins ED-1 and TNF-α were significantly increased in the E and F groups.The expressions of apoptosis-related proteins Bax and cleaved-caspase-3 significantly increased,and the expression of anti-apoptosis-related protein Bcl-2 was significantly decreased.The trend of proteins in group E was more obvious than that of group F(P<0.05).Conlusions1.The iso-osmolar contrast agent can significantly reduce the incidence of CIN than the low-osmolar contrast agent when used in a population of patients with coronary heart disease and chronic congestive heart failure who are currently undergoing coronary intervention procedure.The iso-osmolar contrast agent was associated with a significantly lower nephrotoxicity.Combined detection three new markers of serum cystatinC,urinary NAG enzyme and urinary β2-MG which reflect early renal damage can be more early,comprehensive and accurate assessment of renal damage.2.Contrast agent can inhibit the proliferation of human proximal tubule cells and increase the inflammatory reaction and apoptosis of renal tubular cells.The effect of hypotonic iohexol on cell proliferation,inflammation and apoptosis is more significant than isotonic iodixanol.3.Contrast agent can aggravate mice renal tissue cells and increase inflammation and apoptosis of mice renal tissue.Hypotonic iohexol has more significant damage to mice renal tissue than isotonic iodixanol.更多还原