Joshi Sunil, Wang Wei, Peck Ammon B, Khan Saeed R
Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida.
Department of Infectious Diseases and Pathology, University of Florida College of Veterinary Medicine, Gainesville, Florida.
J Urol. 2015 May;193(5):1684-91. doi: 10.1016/j.juro.2014.11.093. Epub 2014 Nov 28.
We investigated the association of hyperoxaluria and calcium oxalate crystal induced production of reactive oxygen species with activation of the NLRP3 inflammasome.
Eight-week-old male rats were given hydroxy-L-proline to induce hyperoxaluria. A group of rats on the hydroxy-L-proline diet also received apocynin, an antioxidant and nonspecific inhibitor of NADPH oxidase. At 28 days the rats were sacrificed and the kidneys were extracted. Microarray analysis was done with the BeadArray™ Reader. Gene ontology and gene pathway analyses were done with the DAVID (Database for Annotation, Visualization of Integrated Discovery) enrichment analysis tool. Quantitative real-time polymerase chain reaction and immunohistochemical staining were performed to confirm microarray results.
Analysis of 22,226 genes revealed that 20 and 24 pathways were highly significant in the cortex and medulla, respectively. In the cortex extracellular matrix-receptor interaction, complement and coagulation cascades, focal adhesion and hypertrophic cardiomyopathy were the most significant pathways. In the medulla complement and coagulation cascades, extracellular matrix-receptor interaction and dilated cardiomyopathy were the major pathways. Genes encoding for PYCARD (ASC), TXNIP, NLRP3, caspase-1, and IL-1β and 18 were significantly up-regulated in hydroxy-L-proline fed rats but in the group that received apocynin these genes were down-regulated in the cortex and medulla. Results were verified by quantitative real-time polymerase chain reaction with SYBR® Green assay and immunohistochemical staining.
Results indicate a role for reactive oxygen species in activation of the NLRP3 inflammasome via TXNIP. This led to a robust inflammatory response in the kidneys of rats with hyperoxaluria and calcium oxalate nephrolithiasis.
我们研究了高草酸尿症以及草酸钙晶体诱导的活性氧生成与NLRP3炎性小体激活之间的关联。
给8周龄雄性大鼠喂食羟基-L-脯氨酸以诱导高草酸尿症。一组食用羟基-L-脯氨酸饮食的大鼠还接受了载脂蛋白，一种NADPH氧化酶的抗氧化剂和非特异性抑制剂。28天后处死大鼠并取出肾脏。使用BeadArray™ 阅读器进行微阵列分析。使用DAVID（注释、可视化与综合发现数据库）富集分析工具进行基因本体论和基因通路分析。进行定量实时聚合酶链反应和免疫组织化学染色以确认微阵列结果。
对22226个基因的分析表明，分别有20条和24条通路在皮质和髓质中具有高度显著性。在皮质中，细胞外基质-受体相互作用、补体和凝血级联反应、粘着斑和肥厚性心肌病是最显著的通路。在髓质中，补体和凝血级联反应、细胞外基质-受体相互作用和扩张型心肌病是主要通路。在喂食羟基-L-脯氨酸的大鼠中，编码PYCARD（ASC）、TXNIP、NLRP3、caspase-1以及IL-1β和18的基因显著上调，但在接受载脂蛋白的组中，这些基因在皮质和髓质中下调。通过使用SYBR® Green检测的定量实时聚合酶链反应和免疫组织化学染色验证了结果。
结果表明活性氧通过TXNIP在NLRP3炎性小体激活中发挥作用。这在患有高草酸尿症和草酸钙肾结石的大鼠肾脏中引发了强烈的炎症反应。
