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. 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.

患者就诊时的急性肾脏疾病识别仍是临床医学中的核心难题。目前临床仅以单一分析物——血清肌酐（serum creatinine, sCr）作为肾小管、血管或间质细胞损伤的替代标志物，但该检测并非肾损伤特异性指标，反而可能反映远端器官原发疾病引发的生理应答。此外，细胞损伤事件可在数分钟至数小时内发生，但血清肌酐需24小时及以上才能达到临床警戒阈值，或体现组织功能与结构的进一步恶化。本研究改编自Gay等人于2013年发表于《Genes Dev.》的方法（2013, 27(1):98-115, DOI: 10.1101/gad.205278.112），使其可在损伤后按需对特定细胞的RNA进行标记。该技术通过在特定细胞中表达尿嘧啶磷酸核糖转移酶（uracil phosphoribosyltransferase, Uprt），后续纯化4-硫尿嘧啶（4-thio-uracil）标记的新生RNA。本研究以集合管为研究对象，采用该技术发现：两种可升高血清肌酐的模型——血容量减少模型与缺血性损伤模型，并未激活相同的基因簇或基因表达通路。因此，新生RNA的单次检测快照可揭示依赖血清肌酐的诊断分类（即急性肾损伤（AKI））所掩盖的病理复杂性。我们认为，尿嘧啶磷酸核糖转移酶技术可实现在肾单位各细胞类型在损伤后多个时间点的特征分析。本研究数据将以精准医学理念革新现有临床诊断策略。本研究通过Cre重组酶（Cre）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"acute kidney injury"模型（iAKI与vAKI）的转录组特征。