A Snapshot of RNA Expression in a Single Segment of the Kidney Reveals Stimulus Specific Responses

The identification of acute kidney disease at the time of patient encounter remains a central problem in clinical medicine. A single analyte, the serum creatinine (sCr) is currently in use as a surrogate for tubular, vascular, or interstitial cellular damage. Nonetheless the sCr test is not specific to kidney injury, but rather might reflect physiological responses to a the primary disease in a distant organ. In addition, while cellular events occur over minutes or hours, sCr requires 24 hours or more to reach a worrisome clinical threshold or demonstrate further deterioration of tissue function and architecture. Here we have adapted the method of Gay et al, Genes Dev. 2013 27(1):98-115. doi: 10.1101/gad.205278.112. to allow cell specific labelling of RNA at the time of our choosing after an injury. The technique involves the cell specific expression of a uracil phosphoribosyltransferase (Uprt) and the subsequent purification of 4-thio-uracil labelled nascent RNAs. Using this technique focused on the collecting duct, we found that a model of volume depletion and a model of ischemic damage, both of which raise the sCr do not activate the same cohort of genes nor the same pathways of gene expression. Hence, a snapshot of newly synthesized RNA reveals the complexity subsumed by diagnostic classifications dependent on sCr (called 'AKI'). We suggest that the Uprt technique will allow characterization of each cell type in the nephron at multiple time points after the onset of injury. These data will replace our current diagnostic strategies with Precision Medicine. Overall design: Examining transcriptional profiles of two models of "acute kidney injury" (iAKI and vAKI), compared to controls, in a single segment of the kidney using cre mediated 4-thio-Uracil tagging of RNA.

患者就诊时急性肾损伤（acute kidney injury, AKI）的识别仍是临床医学的核心难题。目前临床以单一生物标志物血清肌酐（serum creatinine, sCr）作为肾小管、血管或间质细胞损伤的替代检测指标。然而，血清肌酐检测并非肾损伤特异性指标，反而可能反映远隔器官原发疾病引发的生理应答。此外，尽管细胞损伤事件可在数分钟至数小时内发生，但血清肌酐需要24小时甚至更久才能达到临床关注阈值，或显示出组织功能与结构的进一步恶化。
在此，我们对Gay等人2013年发表于《基因与发育（Genes & Development）》第27卷第1期第98-115页、DOI为10.1101/gad.205278.112的研究方法进行了优化，使其可在损伤后由研究者指定的时间点实现细胞特异性RNA标记。该技术的核心为细胞特异性表达尿嘧啶磷酸核糖转移酶（uracil phosphoribosyltransferase, Uprt），随后纯化被4-硫尿嘧啶（4-thio-uracil）标记的新生RNA。
通过针对集合管（collecting duct）的该技术应用，我们发现：两种均可导致血清肌酐升高的模型——容量耗竭模型与缺血性损伤模型，其所激活的基因集与基因表达通路均不相同。因此，对新生合成RNA的快照式检测，可揭示依赖血清肌酐的诊断分类（即‘急性肾损伤（AKI）’）所涵盖的病理复杂性。
我们认为，尿嘧啶磷酸核糖转移酶技术可实现在损伤发生后的多个时间点对肾单位（nephron）各细胞类型的特征解析，相关数据将助力精准医学（Precision Medicine）取代当前的临床诊断策略。
整体实验设计：通过Cre重组酶介导的4-硫尿嘧啶RNA标记技术，在肾脏单一节段中对比对照组与两种急性肾损伤模型——缺血性AKI（iAKI）与容量性AKI（vAKI）的转录组谱。