【作者】 刘辉；

【导师】 吕永曼；

【作者基本信息】 华中科技大学 ， 内科学（肾病）， 2018， 博士

【摘要】 马兜铃酸肾病(AAN)呈急慢性演化进程。急性马兜铃酸肾病中,马兜铃酸主要损伤近端肾小管上皮细胞(PTEC),肾小管上皮细胞凋亡构成急性AAN发病机制中主要的细胞死亡途径。既往研究证实p53通路参与马兜铃酸诱导的肾小管上皮细胞凋亡过程。同源盒基因Six1是转录调控因子,在细胞发育、增殖、分化、凋亡中发挥作用,同时也参与调控细胞周期,在细胞生长和增殖中起重要作用。已有报道表明Six1可负性调控p53,并且不依赖于p53泛素化降解蛋白MDM2起作用。如果在急性AAN模型中过表达Six1,是否可以通过调控p53减少肾小管上皮细胞凋亡?因此,本课题旨在研究Six1基因通过调控p53通路在急性马兜铃酸肾病中的保护作用及相关机制。首先,在体内实验中,我们采用腹腔注射马兜铃酸(AAI)的方式建立急性马兜铃酸肾病小鼠模型,发现肾小管上皮细胞凋亡增加,同时肾脏组织中Six1的表达减少,p53、p-p53及凋亡相关蛋白cleaved-caspase3(c-caspase3)表达增加。然后运用腺相关病毒(AAV9)肾包膜下局部注射的方式构建肾脏Six1过表达小鼠模型,模型成功后,在此基础上建立急性马兜铃酸肾病小鼠模型。我们观察到,与对照组相比随着Six1的表达水平增加,p53及c-caspase3表达减少,相应肾脏细胞凋亡数目减少,组织损伤减轻。在体外实验则中,我们用10ug/mL马兜铃酸刺激大鼠肾小管上皮细胞(NRK-52E),细胞凋亡明显增加,肾小管上皮细胞Six1表达减少,同时p53、p-p53、c-caspase3表达增加。利用腺病毒过表达技术将肾小管上皮细胞中的Six1基因过表达后,马兜铃酸刺激肾小管上皮细胞表达的p53、p-p53及c-caspase3减少,细胞凋亡减少。综上所述,Six1可下调p53表达,从而抑制凋亡相关通路,最终减少马兜铃酸诱导的肾小管上皮细胞凋亡。因此,上调Six1基因表达水平有望成为临床治疗急性马兜铃酸肾病的新策略。第一部分Six1在急性马兜铃酸肾病中的表达目的:探讨Six1在急性马兜铃酸肾病中的表达变化。方法:首先查询肾脏基因表达数据库Nephromine(https://www.nephroseq.org)明确Six1在人类肾脏疾病中表达变化。然后利用腹腔注射马兜铃酸方式构建小鼠急性马兜铃酸肾病模型,将实验动物分为2组:(1)假手术组(Sham):腹腔注射生理盐水;(2)模型组(AAN):腹腔注射马兜铃酸;予AAI(20mg/kg)一次性腹腔注射诱导急性AAN,分别在AAI注射第1、3、7天处死小鼠,取血清检测肌酐、尿素等肾功能变化,PAS染色观察肾小管损伤程度,免疫组化染色观察Six1表达水平及定位,TUNEL染色观察肾小管上皮细胞凋亡改变。应用Western blot和real-time PCR检测肾脏Six1、p53、p-p53及c-caspase-3等表达变化。结果:CKD患者肾脏中Six1表达明显下降。在急性AAN模型中,与假手术组相比,肾小管中Six1表达明显下降,肾小管上皮细胞凋亡增加,同时肾脏中p53、p-p53及凋亡相关蛋白c-caspase3(cleaved-caspase3)表达增加,Six1表达变化与p53、p-p53及凋亡相关蛋白c-caspase3表达变化呈负相关。结论:在急性马兜铃酸肾病模型中,Six1表达减少伴随肾小管上皮细胞凋亡增加,Six1表达水平异常和马兜铃酸肾病发展有关。第二部分Six1在急性AAN中通过抑制p53介导的PTEC凋亡减轻肾脏损伤目的:探讨Six1在急性马兜铃酸肾病中的保护作用及机制。方法:利用马兜铃酸腹腔注射方式构建急性马兜铃酸肾病小鼠模型,用Six1过表达腺相关病毒(AAV9)肾包膜局部注射的方式构建肾脏Six1过表达小鼠模型,将实验动物分为2组:(1)空载病毒+AAN模型组(Vehicle+AAI):肾包膜下注射空载病毒+马兜铃酸腹腔注射;(2)肾包膜下注射Six1过表达病毒+马兜铃酸腹腔注射。病毒感染21天后予AAI(20mg/kg)一次性腹腔注射诱导急性AAN,分别在AAI注射第1,3,7天处死小鼠,取血清检测肌酐、尿素等肾功能变化,PAS染色观察肾小管损伤程度,TUNEL染色检测肾小管上皮细胞凋亡变化。Real-time PCR检测Six1 m RNA水平改变,Western blot检测肾组织Six1、p53、p-p53及c-caspase-3蛋白表达水平变化。结果:在急性AAN动物模型中,与空载组相比,肾脏Six1过表达组肾小管上皮细胞凋亡数明显减少,肾脏病理损伤减轻,p53、p-p53及凋亡相关蛋白c-caspase-3表达减少,但是两组肾功能改变无统计学差异。结论:在急性马兜铃酸肾病模型中,过表达Six1基因可通过下调p53减轻马兜铃酸诱导的肾小管上皮细胞凋亡而减轻肾脏病理损伤。第三部分Six1在马兜铃酸诱导的肾小管上皮细胞损伤中的作用与机制研究目的:探索Six1在马兜铃酸诱导肾小管上皮细胞损伤中的作用机制。方法:在体外实验中,用AAI刺激大鼠肾小管上皮细胞(NRK-52E),并设置AAI浓度梯度及时间梯度,利用Real-time PCR检测Six1 m RNA水平改变,Western Blot检测Six1和p53、p-p53、c-caspase3的蛋白表达水平。利用过表达Six1腺病毒转染NRK-52E细胞,转染成功后加入AAI刺激,流式细胞术检测细胞凋亡变化,Western blot检测Six1、p53、p-p53、MDM2及凋亡相关蛋白c-caspase-3表达水平。结果:AAI刺激NRK-52E细胞,随着AAI浓度的提高,刺激时间的延长,Six1表达减少,p53及p-p53蛋白表达量逐渐增多;过表达Six1基因后,与空载病毒组相比,AAI刺激诱导的NRK-52E细胞凋亡减少,p53、p-p53、c-caspase-3等蛋白表达减少。与对照组相比,MDM2蛋白水平无变化。结论:Six1可减轻p53介导的AAI诱导的肾小管上皮细胞损伤。更多还原

【Abstract】 Aristolochic acid nephropathy(AAN)is an acute and chronic evolutionary process.In acute aristolochic acid nephropathy,aristolochic acid mainly damages proximal tubular epithelial cells(PTEC),and apoptosis of renal tubular epithelial cells constitutes the major cell death pathway in the pathogenesis of acute AAN.Previous reports have confirmed that p53 pathway is involved in aristolochic acid-induced apoptosis of renal tubular epithelial cells.The homeobox gene Six1 is a transcriptional regulator that plays a role in cell development,proliferation,differentiation,and apoptosis.It also regulates the cell cycle and plays an important role in cell growth and proliferation.It has been reported that Six1 negatively regulates p53 and does not depend on p53 ubiquitination to degrade protein MDM2.If Six1 is overexpressed in the acute AAN model,it can reduce renal tubular epithelial cell apoptosis by regulating p53.Therefore,the purpose of this study is to investigate the protective role of Six1 in the regulation of acute aristolochic acid nephropathy by regulating p53 pathway and related mechanisms.Firstly,we established an animal model of acute aristolochic acid nephropathy by intraperitoneal injection of aristolochic acid(AAI),and found that apoptosis of renal tubular epithelial cells increased,and the expression of Six1 in the kidneys decreased,and the expression of p53,p-p53 and apoptosis-related protein cleaved-caspase3(c-caspase3)increased.We then used the renal subcapsular injection of adeno-associated virus(AAV9)to construct a mouse model of Six1 overexpression in the kidney.After the model was successfully established,a mouse model of acute aristolochic acid nephropathy was established on this basis.We observed that compared with the control group,the expression of Six1 increased,the expression of p53 and c-caspase3 decreased,the corresponding renal cell apoptosis decreased,and the tissue damage was reduced.Then we used a certain concentration of aristolochic acid to stimulate rat renal tubular epithelial cells(NRK-52E)in vitro,apoptosis increased,renal tubular epithelial cells Six1 expression decreased,while the expression of p53,p-p53 and c-caspase3 increased.Using adenovirus overexpression technology to overexpress Six1 gene in renal tubular epithelial cells,aristolochic acid stimulated the reduction of p53,p-p53 and c-caspase3 expression in renal tubular epithelial cells and apoptosis decreased.In summary,Six1 can down-regulate p53,thereby inhibiting apoptosis-related pathways,and ultimately play a role in reducing aristolochic acid-induced apoptosis of renal tubular epithelial cells.Therefore,increasing the expression level of Six1 is expected to become a new strategy for clinical treatment of acute aristolochic acid nephropathy.Part Ⅰ Expression of Six1 in acute aristolochic acid nephropathy Objective: To investigate the expression of Six1 gene in acute aristolochic acid nephropathy.Methods : The Nephromine(https://www.nephroseq.org)database of renal gene expressions was first searched for the expression of Six1 in human kidney disease.Then,the animal model of acute aristolochic acid nephropathy was established by intraperitoneal injection of aristolochic acid.The experimental animals were divided into two groups: 1.sham operation group(sham): saline injection;2.model group(AAN): aristolochic acid injection,AAI(20mg/kg)was injected intraperitoneally to induce acute AAN.The mice were sacrificed on day 1,3,and 7 after AAI injection.Serum levels of creatinine,urea,and other renal function were measured.PAS staining was used to observe the degree of tubule injury,and the expression and localization of Six1 was assessed by immunohistochemical staining.TUNEL staining was used to observe the apoptosis of renal tubular epithelial cells.The expression of Six1,p53,p-p53 and c-caspase-3 in kidneys was detected by Western blot and real-time PCR.Results:The expression of Six1 in kidneys of patients with CKD decreased significantly.In the acute AAN model,compared with the sham group,the expression of Six1 in the renal tubules was significantly decreased,and the apoptosis of renal tubular epithelial cells was increased.At the same time,p53,p-p53 and apoptosis-related protein c-caspase3(cleaved-caspase3)in the kidney.The expression of Six1 was negatively correlated with the expression of p53,p-p53 and apoptosis-associated protein c-caspase3.Conclusion:In the acute aristolochic acid nephropathy model,the decreased expression of Six1 was associated with increased apoptosis of renal tubular epithelial cells and increased renal pathological lesions.Part Ⅱ Six1 alleviates kidney injury by inhibiting p53-mediated PTEC apoptosis in acute AAN Objective:To investigate the protective effect and mechanism of Six1 in acute aristolochic acid nephropathy.Methods : The model of acute aristolochic acid nephropathy was established by intraperitoneal injection of aristolochic acid.The Six1 overexpression mouse kidney model was established by local injection of Six1 overexpressing adeno-associated virus(AAV9)under renal capsule.Two groups: 1 no-load virus+AAN model group(Vehicle+AAI): subrenal injection of no-load virus + aristolochic acid intraperitoneal injection;2 kidney subcapsular injection of Six1 over-expression virus + aristolochic acid intraperitoneal injection.21 days after the virus infection,AAI(20 mg/kg)was intraperitoneally injected to induce acute AAN.The mice were sacrificed on day 1,3,and 7 after AAI injection.Serum levels of creatinine,urea,and other renal functions were measured.PAS staining was performed to observe the injury degree of tubular,TUNEL staining was used to detect the apoptosis of renal tubular epithelial cells.The level of Six1 m RNA was detected by Real-time PCR.The expression of Six1,p53,p-p53 and c-caspase-3 protein in renal tissues was detected by Western blot.Results: In the acute AAN animal model,renal tubular epithelial cell apoptosis and renal pathological lesions reduced in the Six1 overexpression group.Compared with the empty group,the expression p53,p-p53 and apoptosis-associated protein c-caspase-3 decreased,but there was no statistical difference in renal function between the two groups.Conclusion:In the acute aristolochic acid nephropathy model,overexpression of Six1 gene can reduce renal pathological injury by down-regulating p53 and aristolochic acid-induced apoptosis of renal tubular epithelial cells.Part Ⅲ The Role and Mechanism of Six1 in Aristolochic Acid In Renal Tubular Epithelial Cell Injury Objective: To explore the role and mechanism of Six1 in aristolochic acid-induced renal tubular epithelial cell injury.Methods: In vitro,AAI was used to stimulate rat renal tubular epithelial cells(NRK-52E),and AAI concentration gradients and time gradients were set.Real-time PCR was used to detect the expression of Six1 m RNA.Western Blot was used to detect the expression of Six1,p53,p-P53 and c-caspase3 protein.Transfected NRK-52 E cells with adenovirus over-expressing Six1 gene were stimulated with AAI after transfection.Flow cytometry was used to detect apoptosis.Western blot was used to detect the expression of Six1,p53,p-p53,MDM2 and c-caspase-3.Results: With the increase of AAI concentration,the stimulation time prolonged,the expression of Six1 decreased,and the expression of p53 and p-p53 protein gradually increased in NRK-53 E.With the overexpression of Six1 gene,compared with that of no-load virus group.Apoptosis of AAI stimulated NRK-52 E cells was reduced,and the expression of p53,p-p53,c-caspase-3 and other proteins also decreased.There was no difference of MDM2 compared to the control group.Conclusion: Six1 can alleviate p53-mediated AAI-induced renal tubular epithelial cell injury.更多还原