摘要
目的分析不同分期慢性肾脏病(chronic kidney disease,CKD)患者肾组织中发生程序性坏死的肾小管上皮细胞数量及其与临床病理指标的相关性,探讨肾小管上皮细胞程序性坏死在CKD患者肾小管上皮细胞过度死亡、慢性肾损伤进展中的作用。方法收集2017年6月至2019年6月于海南医学院第一附属医院行肾活检且估算肾小球滤过率(eGFR)≥15 ml?min-1?(1.73 m2)-1的60例18~65岁CKD患者的临床资料和肾组织活检标本。按照肾脏病预后质量指南(K/DOQI)分为CKD 1~4期,每期15例,以不伴有基础肾脏疾病的肾外伤患者为对照(n=15)。采用原位末端转移酶标记技术(TUNEL)+受体相互作用蛋白3(receptor-interacting protein 3,RIP3)免疫荧光、免疫组化技术分别检测不同时期CKD患者肾组织中发生程序性坏死的肾小管上皮细胞数和程序性坏死标志性蛋白RIP3、混合系激酶区域样蛋白(mixed lineage kinase domain-like protein,MLKL)的表达,并比较其差异。采用Pearson相关分析分析肾组织中程序性坏死肾小管上皮细胞百分数与临床病理指标的相关性。同时,也分析肾组织血管紧张素Ⅱ2型受体(AT2R)表达及其与程序性坏死肾小管上皮细胞百分数的相关性。结果按CKD分期比较结果显示,随着CKD进展,CKD患者肾小管的结构破坏逐渐加重,相应区域的肾小管萎缩,伴间质纤维化;相邻区域肾小管灶性扩张、肾小管内可见大量蛋白管形。CKD 2、3期患者肾小管损伤分数明显高于对照组(均P<0.01);TUNEL+RIP3免疫荧光染色结果显示,CKD 2、3期患者肾小管上皮细胞TUNEL、RIP3双阳性肾小管上皮细胞(程序性坏死细胞)百分数较高(均P<0.01)。免疫组化结果显示,CKD患者肾组织RIP3、MLKL和AT2R蛋白主要表达于肾小管上皮细胞胞质内,在CKD 2、3期患者肾小管上皮细胞胞质表达较高(均P<0.05)。Pearson相关分析结果显示,肾小管上皮细胞程序性坏死百分数与尿素氮(r=0.514,P=0.003)、血肌酐(r=0.507,P=0.019)、胱抑素C(r=0.571,P=0.026)、血尿酸(r=0.592,P=0.008)、肾小管损伤分数(r=0.901,P<0.001)、肾间质纤维化指数(r=0.700,P=0.001)及肾组织AT2R蛋白表达(r=0.715,P=0.001)呈正相关。结论在CKD进展中,患者肾小管上皮细胞发生了程序性坏死。程序性坏死可能是导致CKD患者肾小管上皮细胞过度死亡、慢性肾损伤不断进展的重要因素,并且肾小管上皮细胞程序性坏死可能与肾组织中高表达的AT2R相关。
Objective To investigate the number of necroptotic renal tubular epithelial cells in renal tissues of patients with chronic kidney disease(CKD)and the correlation with clinicopathologic parameters,and explore its role in the progression of the excessive loss of renal tubular cells and chronic kidney injury.Methods Renal tissue samples from 60 patients(18-65 years old)with CKD proven by kidney biopsy in the First Affiliated Hospital of Hainan Medical University from June 2017 to June 2019 were collected.According to internationally accepted K/DOQI guidelines,the patients were divided into 1-4 stages of CKD,with 15 cases in each stage.The number of necroptotic renal tubular epithelial cells in patients with different stages of CKD was detected using receptor-interacting protein 3(RIP3)and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)fluorescent staining,and the expression of RIP3 and MLKL,marker protein of necroptosis,was detected by immunohistochemistry.Pearson correlation analysis was used to analyze the correlation between the percentage of necroptotic renal tubular epithelial cells and clinicopathologic parameters.In addition,the expression of angiotensinogenⅡreceptor(AT2R)in renal tissue and its correlation with the percentage of necroptotic renal tubular epithelial cells were analyzed.Results With the development of CKD,the structural destruction of renal tubules in patients with CKD was gradually aggravated,and the renal tubules in the corresponding areas were atrophied,accompanied by worsening interstitial fibrosis.The adjacent renal tubules were focally dilated and numerous protein tubules were seen in the tubules.Importantly,renal tubular injury score in second and third stage of CKD was significantly higher than that in control group(both P<0.01).TUNEL+RIP3 immunofluorescence staining results showed that the percentage of TUNEL/RIP3 double positive renal tubular epithelial cells(necroptotic renal tubular epithelial cells)in renal tubules of the second and third stage of CKD was higher(all P<0.01).Immunohistochemical results showed that RIP3,MLKL and AT2R proteins were mainly expressed in cytoplasm of renal tubular epithelial cells,and the expression of RIP3,MLKL and AT2R in renal tubular epithelial cells was higher in the second and third stage of CKD patients(all P<0.05).Pearson correlation analysis showed that the percentage of necroptotic renal tubular epithelial cells was positively correlated with blood urea nitrogen(r=0.514,P=0.003),serum creatinine(r=0.507,P=0.019),serum cystatin C(r=0.571,P=0.026),serum uric acid(r=0.592,P=0.008),renal tubules injury score(r=0.901,P<0.001),renal interstitial fibrosis index(r=0.700,P=0.001)and the expression of AT2R protein in renal tissue(r=0.715,P=0.001).Conclusions As CKD progresses,necroptosis of renal tubular epithelial cells in CKD patients occurs.The necroptotic cell death may be an important factor leading to renal tubular epithelial cell excessive death and the progression of chronic kidney injury.Furthermore,necroptosis of renal tubular epithelial cells may be related to the high expression of AT2R in kidney tissue.
作者
朱永俊
李晓燕
吕潇阳
王善志
沈婕
林子艳
钟良宝
Zhu Yongjun;Li Xiaoyan;Lyu Xiaoyang;Wang Shanzhi;Shen Jie;Lin Ziyan;Zhong Liangbao(Department of Nephrology,the First Affiliated Hospital of Hainan Medical University,Haikou 570100,China)
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2021年第1期23-30,共8页
Chinese Journal of Nephrology
基金
国家自然科学基金(81660131)
海南省重点研发计划科技合作方向项目(ZDYF2019215)。
