利用 CRISPR/Cas9 系统在大鼠中生成 1 型原发性高草酸尿症疾病模型。
Generation of a Primary Hyperoxaluria Type 1 Disease Model Via CRISPR/Cas9 System in Rats.
机构信息
Department of Pediatric Urology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
出版信息
Curr Mol Med. 2018;18(7):436-447. doi: 10.2174/1566524019666181212092440.
BACKGROUND
Primary hyperoxaluria type 1 (PH1) is an inherited disease caused by mutations in alanine-glyoxylate aminotransferase (AGXT). It is characterized by abnormal metabolism of glyoxylic acid in the liver leading to endogenous oxalate overproduction and deposition of oxalate in multiple organs, mainly the kidney. Patients of PH1 often suffer from recurrent urinary tract stones, and finally renal failure. There is no effective treatment other than combined liver-kidney transplantation.
METHODS
Microinjection was administered to PH1 rats. Urine samples were collected for urine analysis. Kidney tissues were for Western blotting, quantitative PCR, AGT assays and histological evaluation.
RESULTS
In this study, we generated a novel PH1 disease model through CRISPR/Cas9 mediated disruption of mitochondrial localized Agxt gene isoform in rats. Agxt-deficient rats excreted more oxalate in the urine than WT animals. Meanwhile, mutant rats exhibited crystalluria and showed a slight dilatation of renal tubules with mild fibrosis in the kidney. When supplied with 0.4% ethylene glycol (EG) in drinking water, mutant rats excreted greater abundance of oxalate and developed severe nephrocalcinosis in contrast to WT animals. Significantly elevated expression of inflammation- and fibrosisrelated genes was also detected in mutants.
CONCLUSION
These data suggest that Agxt-deficiency in mitochondria impairs glyoxylic acid metabolism and leads to PH1 in rats. This rat strain would not only be a useful model for the study of the pathogenesis and pathology of PH1 but also a valuable tool for the development and evaluation of innovative drugs and therapeutics.
背景
原发性高草酸尿症 1 型（PH1）是一种遗传性疾病，由丙氨酸-乙醛酸氨基转移酶（AGXT）基因突变引起。其特征为肝脏中乙醛酸代谢异常，导致内源性草酸生成过多，并在多个器官，主要是肾脏中沉积草酸。PH1 患者常反复发作尿路结石，最终导致肾功能衰竭。除了联合肝肾移植外，目前尚无有效治疗方法。
方法
对 PH1 大鼠进行微注射。收集尿液样本进行尿液分析。取肾组织进行 Western blot、定量 PCR、AGT 测定和组织学评估。
结果
在这项研究中，我们通过 CRISPR/Cas9 介导的大鼠线粒体定位 Agxt 基因亚型的破坏，产生了一种新型 PH1 疾病模型。Agxt 缺陷型大鼠尿液中排泄的草酸多于 WT 动物。同时，突变型大鼠出现结晶尿，并在肾脏中表现出轻微的肾小管扩张和轻度纤维化。当给予 0.4%乙二醇（EG）饮用水时，突变型大鼠排泄出更多的草酸，并与 WT 动物相比发展出严重的肾钙质沉着症。在突变体中还检测到炎症和纤维化相关基因的显著上调。
结论
这些数据表明，线粒体中 Agxt 的缺失会损害乙醛酸代谢，导致大鼠发生 PH1。这种大鼠品系不仅将成为研究 PH1 发病机制和病理学的有用模型，而且将成为开发和评估创新药物和治疗方法的有价值工具。
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