【作者】 张波；

【导师】 陈湘；

【作者基本信息】 中南大学 ， 临床医学（专业学位）， 2023， 博士

【摘要】 研究背景:梗阻性肾病是一种因尿液排出障碍而导致肾脏结构和功能损害的疾病,其最显著的病理特征是肾纤维化。导致梗阻性肾病的病因包括泌尿系统结石、畸形、肿瘤及前列腺增生等。研究发现梗阻性肾病是一种慢性进展性和难逆转性疾病,部分已发生中重度积水的肾脏即使在梗阻解除后其功能及结构仍然难以恢复,纤维化进展仍在继续。目前尚无确切药物可以逆转或终止肾纤维化进展,如何减轻梗阻引起的肾脏损伤,促进解除梗阻后肾脏结构及功能恢复是临床亟待解决的难题。深入研究梗阻性肾病肾纤维化发生机制,探讨新的抗肾纤维化靶点是关键切入点。铁死亡是一种新型细胞死亡方式,本质是铁依赖性的程序性细胞死亡,由过度的脂质过氧化物引起。肾小管上皮细胞是肾脏损伤时的最初反应者,其发生损伤被认为是肾纤维化的起始环节。目前已证实铁死亡与多种疾病的发生发展密切相关,铁死亡是急性肾损伤时肾小管上皮细胞重要的死亡方式,但慢性肾纤维化过程中是否发生肾小管上皮细胞铁死亡及铁死亡与慢性肾纤维化之间的联系尚不清楚。Liproxstatin-1(Lip-1)是一种有效的铁死亡抑制剂,且不干扰其他经典类型的细胞死亡,但Lip-1在肾纤维化中的作用尚未见文献报道。目的:本研究拟:(1)收集临床梗阻性肾病肾组织样本并构建单侧输尿管梗阻(unilateral ureteral obstruction,UUO)小鼠肾纤维化模型探讨梗阻性肾纤维化中肾小管上皮细胞是否发生铁死亡;(2)在整体水平探讨Lip-1对UUO模型中肾小管上皮细胞铁死亡的作用及对肾纤维化程度的影响及其机制;(3)在细胞水平探讨Lip-1减轻梗阻性肾纤维化的细胞及分子机制。方法与结果:1.梗阻性肾纤维化组织中肾小管上皮细胞发生铁死亡1.1梗阻性肾病肾组织发生纤维化并可能存在肾小管上皮细胞铁死亡收集人体梗阻性肾病肾组织和正常肾组织(远离肿瘤的相对正常肾组织),HE染色和Masson染色检测肾脏组织病理学改变及胶原沉积情况。结果显示,梗阻性肾病肾组织发生病理性结构破坏,胶原沉积明显增加。普鲁士蓝染色以及免疫荧光技术检测谷胱甘肽过氧化物酶4(GPX4)与E-cadherin(肾小管上皮细胞标志物)共标情况,反映肾组织中肾小管上皮细胞铁死亡情况。结果显示,与正常肾组织相比,梗阻性肾病肾组织中铁沉积增加,铁染色阳性细胞比例明显增多,E-cadherin~+肾小管上皮细胞中GPX4水平明显降低。1.2 UUO模型小鼠肾组织中肾小管上皮细胞发生铁死亡采用UUO法构建小鼠梗阻性肾纤维化模型。于造模后14天收集肾组织,对肾组织进行普鲁士蓝染色、检测丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性、免疫组织化学法检测铁死亡关键分子胱氨酸/谷氨酸逆向转运体轻链亚基(x CT)和GPX4水平、免疫荧光技术检测GPX4与E-cadherin共标情况、免疫荧光技术检测TUNEL与E-cadherin共标情况反映对照组(sham组)及实验组(UUO组)中肾小管上皮细胞铁死亡情况。结果显示,与sham组相比,UUO组肾组织中铁染色阳性细胞比例明显升高,MDA含量明显增高,SOD活性明显降低,x CT和GPX4水平明显降低,UUO组中E-cadherin~+肾小管上皮细胞中GPX4水平降低且TUNEL染色呈阳性的细胞比例明显增加。此外,通过分析GEO数据库中高通量测序数据反映铁死亡相关基因的表达变化,结果显示UUO组肾组织中与脂质代谢途径、铁代谢途径、谷胱甘肽途径、铁自噬途径相关的部分重要基因表达发生明显变化。以上结果表明,UUO诱导的肾纤维化模型中肾小管上皮细胞发生了铁死亡。2.Lip-1减轻UUO诱导的肾小管上皮细胞铁死亡及肾纤维化2.1 Lip-1减轻UUO模型中肾小管上皮细胞铁死亡将小鼠随机分为4组:sham组、Lip-1组、UUO组和UUO+Lip-1组,于造模14天后收集肾组织及血清。检测sham组和Lip-1组肾重/体重比,血清肾功能和肾脏组织结构反映Lip-1对正常肾脏结构和肾功能的影响。结果显示,10 mg/kg/d的Lip-1对正常肾脏组织结构、肾重/体重比和肾功能无明显影响。检测sham组,UUO组和UUO+Lip-1组肾组织中铁含量、MDA含量、SOD活性、氧化型谷胱甘肽(GSSG)/还原型谷胱甘肽(r GSH)比值、GPX4蛋白水平和细胞死亡情况反映Lip-1对肾小管上皮细胞铁死亡的影响。结果显示,Lip-1可以明显抑制UUO模型肾组织中铁含量增加,抑制MDA含量的增加并提高SOD活性,抑制GSSG/r GSH比值的升高,明显上调肾小管上皮细胞中GPX4的蛋白水平并抑制肾小管上皮细胞死亡。以上结果表明,Lip-1可减少UUO模型肾组织中铁含量,减轻脂质过氧化及氧化应激水平,抑制肾小管上皮细胞中GPX4蛋白水平的降低及死亡情况,说明Lip-1可抑制UUO模型肾组织中肾小管上皮细胞铁死亡。2.2 Lip-1减轻UUO诱导的肾纤维化程度通过检测sham组,UUO组和UUO+Lip-1组肾重/体重比、血清肾功能、肾组织形态结构、肾脏间质胶原沉积情况、胶原蛋白I水平反映Lip-1对肾纤维化程度的影响。结果显示,Lip-1可以减轻UUO诱导的小鼠肾重/体重比、血肌酐及尿素氮的增高,改善肾组织形态结构破坏,减轻肾脏间质胶原沉积和胶原蛋白I的水平。通过检测肾组织中促纤维化因子转化生长因子β1(TGF-β1)、结缔组织生长因子(CTGF)和血小板衍生生长因子(PDGF)水平,成纤维细胞增殖指标增殖细胞核抗原(PCNA)及肌成纤维细胞标志物α-平滑肌肌动蛋白(α-SMA)水平探讨Lip-1减轻肾纤维化的机制。结果显示,Lip-1可以降低UUO诱导的肾纤维化组织中促纤维化因子TGF-β1、CTGF、PDGF的水平,降低PCNA和α-SMA水平。以上结果表明,Lip-1可以减轻UUO诱导的肾纤维化程度,其机制可能与降低促纤维化因子水平,抑制成纤维细胞增殖和分化相关。3.Lip-1减轻UUO所致肾纤维化的细胞分子机制3.1 Lip-1抑制肾小管上皮细胞铁死亡使用铁死亡诱导剂RSL3刺激和敲低GPX4(shGPX4)分别诱导肾小管上皮细胞(HK2)铁死亡。通过检测细胞活力、细胞中铁含量、MDA含量变化反映Lip-1对HK2铁死亡的影响。结果显示,Lip-1可以明显改善RSL3或shGPX4诱导的细胞活力下降,减少细胞中铁含量并抑制MDA增高。该结果表明Lip-1可以抑制RSL3和shGPX4诱导的HK2细胞铁死亡。3.2 Lip-1抑制RSL3或shGPX4处理的HK2细胞培养液对成纤维细胞的促增殖和分化作用为明确HK2细胞发生铁死亡对成纤维细胞的影响,收集RSL3或shGPX4处理的HK2细胞条件培养液处理成纤维细胞。CCK8法检测成纤维细胞的增殖,Western Blot检测α-SMA水平反映成纤维细胞分化。结果显示,与control组相比,RSL3或shGPX4诱导HK2细胞铁死亡的细胞培养液可以促进成纤维细胞增殖及向肌成纤维细胞的分化,而经Lip-1处理的条件培养液可以抑制这一过程。以上结果表明HK2细胞铁死亡后释放的物质可以促进成纤维细胞增殖及向肌成纤维细胞的分化,Lip-1通过抑制HK2细胞铁死亡从而有效减轻细胞条件培养液对成纤维细胞的促增殖及分化作用。3.3 Lip-1通过抑制HK2细胞铁死亡减少促纤维化因子的分泌为进一步探讨HK2细胞发生铁死亡介导成纤维细胞增殖及向肌成纤维细胞分化的机制,采用ELISA法检测各组HK2细胞培养液中促纤维化因子TGF-β1、CTGF和PDGF的含量。结果显示,RSL3刺激或shGPX4诱导HK2细胞铁死亡后细胞培养液中TGF-β1、CTGF和PDGF水平明显升高,Lip-1处理显著减少RSL3刺激或shGPX4处理的HK2细胞所释放的促纤维化因子水平。以上结果表明Lip-1通过减少RSL3或shGPX4诱导的HK2细胞铁死亡,减少其分泌促纤维化因子,可减轻其对成纤维细胞的促增殖与分化作用,这可能是Lip-1在整体水平减轻UUO诱导的肾纤维化的重要细胞分子机制。结论:1.人体梗阻性肾病肾纤维化组织及UUO诱导的肾纤维化组织中肾小管上皮细胞发生铁死亡。2.Lip-1可减轻UUO诱导的肾小管上皮细胞铁死亡和肾纤维化程度,Lip-1减轻肾纤维化的机制与降低肾组织中促纤维化因子水平和抑制成纤维细胞增殖及分化相关。3.Lip-1可在细胞水平减轻RSL3刺激或敲低GPX4诱导的HK2细胞铁死亡,减少促纤维化因子的分泌。4.Lip-1通过减轻HK2细胞铁死亡,减少促纤维化因子的分泌而抑制成纤维细胞的增殖与分化,这可能是Lip-1减轻UUO所致肾纤维化的重要细胞分子机制。图38幅,表15个,参考文献102篇更多还原

【Abstract】 Background:Obstructive nephropathy is a common urologic disease in which the structure and function of the kidneys are damaged due to obstruction of urine excretion,and its most prominent pathological feature is renal fibrosis.The causes of obstructive nephropathy include urinary stones,deformities,tumors,and benign prostatic hyperplasia,etc.Studies have found that obstructive nephropathy is a chronic progressive and irreversible disease.After the obstruction is removed,renal function and renal structure are still difficult to recover,and the progress of fibrosis continues.At present,there is no drug can reverse or stop the progression of renal fibrosis.How to reduce the renal damage caused by obstruction and promote the recovery of renal structure and function after the obstruction is relieved is a clinical puzzle that needs to be solved urgently.Studying the mechanism of occurrence and development of renal fibrosis in obstructive nephropathy and exploring new anti-renal fibrosis targets are the key points.Ferroptosis is a newly type of regulated cell death characterized by iron-dependent and excessive lipid peroxidation.Renal tubular epithelial cells(TECs)are the initial responders to renal injury,and their injury is considered to be the initial link of renal fibrosis.It has been confirmed that ferroptosis is closely related to the occurrence and development of various diseases,and ferroptosis is an important death method of TECs during acute kidney injury.However,the link between ferroptosis and chronic renal fibrosis is still unclear.Liproxstatin-1(Lip-1)is a potent ferroptosis inhibitor and does not interfere with other classical types of cell death,but the role of Lip-1 in renal fibrosis has not been reported.Objective:This study aim to:(1)collect renal tissue samples from patients with clinical obstructive nephropathy and construct unilateral ureteral obstruction(UUO)mouse model to verify whether TECs undergo ferroptosis in obstructive renal fibrosis;(2)investigate the effect of Lip-1on ferroptosis of TECs in the UUO model and its effect on the degree of renal fibrosis and its mechanism;(3)explore the cellular and molecular mechanism of Lip-1 on renal fibrosis in vitro.Methods and Results:1.Ferroptosis of TECs in obstructive renal fibrosis1.1 Fibrosis and ferroptosis in renal tissue of obstructive nephropathyHuman obstructive nephropathy renal tissue and relatively normal renal tissue(far away from the tumor)were collected.HE staining and Masson staining were used to detect renal histopathological changes and collagen deposition.The results showed that the renal tissue of obstructive nephropathy had pathological structural damage,and collagen deposition was significantly increased.Perl’s staining and the level of glutathione peroxidase 4(GPX4)and E-cadherin(a marker of TECs)detected by immunohistochemistry in renal tissue reflected the ferroptosis of TECs in renal tissue.The results showed that compared with normal kidney tissue,iron deposition was increased in obstructive nephropathy renal tissue,the number of iron-positive cells was significantly increased,and the level of GPX4 in TECs was significantly decreased.1.2 Ferroptosis of TECs in the kidney tissue of UUO modelRenal fibrosis model was established by UUO method.Kidney tissues were collected 14 days after modeling.Perl’s staining,malondialdehyde(MDA)content and superoxide dismutase(SOD)activity,the levels of ferroptosis key molecules x CT and GPX4 detected by immunohistochemistry,the co-labeling of GPX4 and E-cadherin detected by immunofluorescence,and the co-labeling of TUNEL and E-cadherin detected by immunofluorescence,were used to reflecte the ferroptosis of TECs in the sham group and UUO group.The results showed that compared with the sham group,the number of iron-positive cells in the renal tissue of the UUO group was significantly increased,the content of MDA was significantly increased,the activity of SOD was significantly decreased,and the expressions of x CT and GPX4 were significantly decreased.The number of TUNEL~+cells in E-cadherin~+TECs increased significantly.In addition,by analyzing the high-throughput sequencing data in the GEO database,the expression changes of ferroptosis-related genes were reflected.The results showed that some important gene expression related to lipid metabolism pathway,iron metabolism pathway,glutathione pathway and iron autophagy pathway significant changed.These results indicated that ferroptosis occurred in TECs in UUO-induced renal fibrosis model.2.Lip-1 alleviates UUO-induced ferroptosis of TECs and renal fibrosis2.1 Lip-1 attenuates UUO-induced ferroptosis of TECsC57BL/6 mice were randomly divided into the following four groups:sham group,Lip-1 group,UUO group,and UUO+Lip-1 group.Kidney tissue and serum were collected 14 days after modeling.The kidney weight/body weight ratio,serum renal function and renal tissue structure in sham group and Lip-1 group were detected to reflect the effect of Lip-1 on normal kidney structure and renal function.The results showed that Lip-1 at 10 mg/kg/d had no significant effect on normal kidney tissue structure,kidney weight/body weight ratio and renal function.Iron content,MDA content,SOD activity,oxidized glutathione(GSSG)/reduced glutathione(r GSH)ratio,GPX4 protein level and cellular death situation reflected the effect of Lip-1 on ferroptosis of TECs.The results showed that Lip-1 could significantly inhibit the increase of iron content in UUO model renal tissue,inhibit the increase of MDA content and increase the activity of SOD,inhibit the increase of GSSG/r GSH ratio,significantly up-regulate the protein level of GPX4 in renal tubular epithelial cells,and inhibit TECs death.The above results showed that Lip-1 could reduce iron deposition,cell death,lipid peroxidation,and inhibited the downregulation of GPX4 expression induced by UUO,ultimately inhibiting ferroptosis in TECs.2.2 Lip-1 alleviates UUO-induced renal fibrosisTo explore the effects of Lip-1 on the degree of renal fibrosis.Kidney weight/body weight ratio,serum renal function,renal tissue morphological structure,interstitial collagen deposition,and collagen I level were detected in sham group,UUO group and UUO+Lip-1 group.The results showed that Lip-1 could reduce the UUO-induced increase of kidney weight/body weight ratio,serum creatinine and blood urea nitrogen in mice,improve the morphological and structural damage of kidney tissue,and significantly reduce renal interstitial collagen deposition and collagen I levels.By detecting the levels of the profibrotic factor transforming growth factorβ1(TGF-β1),connective tissue growth factor(CTGF)and platelet-derived growth factor(PDGF)in renal tissue,fibroblast proliferation indicators proliferating cell nuclear antigen(PCNA)and the level of fibroblast markerα-smooth muscle actin(α-SMA),we could investigate the mechanism of Lip-1 alleviating renal fibrosis.The results showed that Lip-1 could inhibit the expression of TGF-β1,CTGF and PDGF in UUO-induced renal fibrosis,and reduce the levels of PCNA andα-SMA.The above results indicate that Lip-1 can alleviate the degree of UUO-induced renal fibrosis,and its mechanism may be related to inhibiting the expression of profibrotic factors and inhibiting the proliferation and differentiation of fibroblasts.3.The cellular and molecular mechanism of Lip-1 alleviating UUO-induced renal fibrosis3.1 Lip-1 inhibits ferroptosis in HK2 cellsIn this study,we used RSL3 treatment or knockdown of GPX4 level(shGPX4)in HK-2 cells to induce ferroptosis.Detecting the changes of cell viability,iron content in cells and MDA content.The results showed that Lip-1 could significantly improve the decrease of cell viability induced by RSL3 or shGPX4,reduce the iron content in cells and inhibit the increase of MDA.This result indicated that Lip-1 could inhibit RSL3or shGPX4-induced ferroptosis in HK2 cells.3.2 Lip-1 inhibits the proliferation and differentiation of fibroblasts treated with HK2 cell culture mediumTo investigate the interactions between TECs and surrounding fibroblasts,we collected RSL3 and shGPX4-induced HK2-conditioned medium(CM)and co-cultured with fibroblasts.The proliferation of fibroblasts was detected by CCK8 assays,and the level ofα-SMA was detected by Western Blot to reflect the differentiation of fibroblasts.Our findings indicated that CM from RSL3 and shGPX4-treated HK2 cells significantly increased fibroblast cell proliferation and enhanced the expression ofα-SMA in fibroblasts,while the CM treated with Lip-1could inhibit this effect.The above results indicate that the substances released after ferroptosis of HK2 cells can promote the proliferation of fibroblasts and fibroblast-to-myofibroblast differentiation,and Lip-1 can effectively reduce the effect by inhibiting the ferroptosis of HK2 cells induced by RSL3 or shGPX4.3.3 Lip-1 reduces the secretion of profibrotic factors by inhibiting RSL3or shGPX4-induced ferroptosis in HK2 cellsTo further explore the mechanism of HK2 cells ferroptosis-mediated fibroblast proliferation and myofibroblast differentiation,we detected the contents of profibrotic factors TGF-β1,CTGF and PDGF in the CM of HK2 cells by ELISA.The results showed that the levels of TGF-β1,CTGF and PDGF in the HK2-CM were significantly increased after RSL3 or shGPX4-induced ferroptosis,and Lip-1 treatment significantly reduced the levels of profibrotic factors.The above results indicate that Lip-1 can reduce the proliferation and differentiation of fibroblasts by reducing the ferroptosis of HK2 cells and the secretion of profibrotic factors.Conclusion:1.Ferroptosis occurres in TECs in human obstructive nephropathy renal fibrosis tissue and UUO-induced renal fibrosis tissue.2.Lip-1 can alleviate UUO-induced ferroptosis and renal fibrosis in TECs,and its mechanism is related to inhibiting the level of profibrotic factors in renal tissue and inhibiting the proliferation and differentiation of fibroblasts.3.Lip-1 can alleviate RSL3-induced and GPX4 knockdown-induced ferroptosis as well as the secretion of profibrotic factors in HK2 cells.4.Lip-1 alleviates HK2 ferroptosis as well as the expression of profibrotic factors,thereby indirectly inhibiting the proliferation and activation of fibroblasts.This study is expected to provide insight into a new approach and new mechanism for the treatment of UUO-induced renal fibrosis.更多还原