草酸钙结晶肾损伤小鼠肾脏转录组学和蛋白组学联合分析

Joint analysis of renal transcriptomics and proteomics in calcium oxalate crystal kidney injury mice

目的了解草酸钙结晶肾损伤过程中肾组织基因及蛋白的动态变化,探讨草酸钙结晶肾损伤的可能机制。方法采用10只8周龄雄性C57BL/6J小鼠,适应性饲养1周,按随机数字表法将其分为对照组和草酸钙结晶肾损伤组(模型组),应用乙醛酸(50%,9 mmol/L)腹腔注射(100 mg·kg-1·d-1,持续5 d)建立草酸钙结晶肾损伤小鼠模型,对照组小鼠予等体积0.9%氯化钠溶液腹腔注射。使用HE染色、PAS染色、Masson染色及Von Kossa染色观察模型组小鼠是否造模成功。对小鼠肾组织进行转录组学和蛋白组学测序,根据差异基因和差异蛋白结果筛选与草酸钙结晶肾损伤有关的基因和蛋白并进行基因本体(gene ontology,GO)及京都基因和基因组数据库(Kyoto Encyclopedia of Genes and Genomes,KEGG)分析。采用主成分分析图、热图及火山图进行肾组织转录组学和蛋白组学分析。采用韦恩图分析差异基因和差异蛋白的重叠内容。结果模型组小鼠肾脏HE染色、PAS染色结果显示肾组织形态结构异常,Masson染色结果显示肾组织存在纤维化,Von Kossa染色结果显示皮髓交界处大量钙盐沉积,血肌酐、血尿素氮等肾损伤标志物显著升高,提示草酸钙结晶肾损伤小鼠模型构建成功。转录组学分析结果显示,相比对照组,模型组共存在2815个显著差异基因,其中2004个基因上调,811个基因下调;蛋白组学分析结果显示,模型组共存在1197个差异蛋白,其中353个蛋白上调,844个蛋白下调。共有338个差异基因与差异蛋白相对应,其中324个差异基因与差异蛋白趋势一致。在表达上调及下调对应的差异基因和差异蛋白中均选取表达差异较大的5个差异分子,分别为血清淀粉样蛋白A1(SAA1)、微小染色体维持蛋白4(MCM4)、精氨酸酶2(ARG2)、S100钙结合蛋白A6(S100A6)、白细胞分化抗原14(CD14)、葡萄糖-6-磷酸酶催化亚基(G6PC)、溶质转运家族22成员6(SLC22A6)、溶质载体有机阴离子转运蛋白家族成员1A1(SLCO1A1)、磷酸烯醇丙酮酸羧激酶1(PEPCK1)和吲哚胺N-甲基转移酶(INMT)。GO分析表明2个组学相关的差异分子富集的共同通路主要涉及线粒体功能、多种能量代谢通路及免疫失调;而KEGG富集分析显示2个组学相关的差异分子主要的富集通路包括转运体异常、线粒体功能障碍、神经退行性病变、氨基酸代谢通路异常、氧化磷酸化等能量代谢通路异常等。结论草酸钙结晶肾损伤小鼠肾脏基因和蛋白层面均发生明显变化。线粒体功能障碍、多种能量代谢通路异常及免疫失调可能作为重要机制参与草酸钙结晶肾损伤的发病。

Objective To understand the dynamic changes of genes and proteins in renal tissues of calcium oxalate crystal kidney injury,and to explore the possible mechanism of calcium oxalate crystal kidney injury.Methods Ten 8-week-old male C57BL/6J mice were adaptively fed for 1 week and divided into control group and calcium oxalate crystal kidney injury group(model group)according to random number table method.The calcium oxalate crystal kidney injury mice were established by intraperitoneal injection of glyoxylate(50%,9 mmol/L,100 mg·kg-1·d-1 for 5 days).Mice in control group were intraperitoneally injected with 0.9%sodium chloride solution of equal volume.HE staining,PAS staining,Masson staining,and Von Kossa staining were used to observe whether the model mice were successfully constructed.Transcriptome and proteome sequencing were performed on the kidneys of control mice and model mice.Genes and proteins related to calcium oxalate crystal kidney injury were screened according to the results of differential expressed genes(DEGs)and differential abundance proteins(DAPs),and gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were performed based on the above results.Principal component analysis,heat map and volcano map were used to perform the transcriptomic and proteomic analysis of renal tissues.Venn diagram was used to analyze the overlap of differential genes and differential proteins.Results Von Kossa staining of the kidney showed a large amount of calcium salt deposition at the cuticer-pulp junction,and serum creatinine,blood urea nitrogen and other markers of kidney injury significantly increased in the model group,suggesting that calcium oxalate crystal kidney injury model was established successfully.Transcriptome and proteome sequencing showed that,compared with control group,2815 DEGs and 1197 DAPs were obtained in the model group,respectively;2004 DEGs and 353 DAPs were significantly up-regulated;811 DEGs and 844 DAPs were significantly down-regulated in the model group.A total of 338 DEGs corresponded with DAPs,324 of which had the same trend with DAPs.The significantly different 10 molecules included serum amyloid A1(SAA1),minichromosome maintenance 4(MCM4),arginase 2(ARG2),S100 calcium-binding protein A6(S100A6),cluster of differentiation 14(CD14),glucose-6-phosphatase catalytic subunit(G6PC),solute carrier family 22 member 6(SLC22A6),solute carrier organic anion transporter family member 1A1(SLCO1A1),phosphoenolpyruvate carboxykinase 1(PEPCK1)and indolamine N-methyltransferase(INMT)in the up-regulated and down-regulated differential genes and proteins.GO analysis indicated that the commonly enriched pathways in the two groups of correlated differential molecules were primarily involved in mitochondrial dysfunction,energy metabolism pathways and immune disorder.KEGG enrichment analysis showed that the differentially expressed molecules were mainly enriched in transporter anomaly,mitochondrial dysfunction,neurodegenerative diseases,amino acid metabolism and abnormal energy metabolism pathways such as oxidative phosphorylation.Conclusions Abundant genes and proteins changes in kidneys of calcium oxalate crystal kidney injury mice.Mitochondrial dysfunction,multiple metabolic pathways abnormalities,and immune response may be important mechanisms involved in the pathogenesis of calcium oxalate crystal kidney injury.