Extracellular RNA moves from the glomerulus to the renal tubule
Extracellular RNA moves from the glomerulus to the renal tubule
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DOI：
10.1101/2021.06.15.448584
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发表时间：
2021-06
期刊：
影响因子：
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通讯作者：
R. Hunter;Sujai Kumar;R. Coward;A. Buck;J. Dear
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文献类型：
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作者：
R. Hunter;Sujai Kumar;R. Coward;A. Buck;J. Dear
有大量的间接证据表明，细胞外RNA（exRNA）信号可以调节肾小管上皮细胞的功能。然而，这种信号的生理重要性是不确定的。我们试图确定健康肾脏细胞间胞外RNA转移的程度。我们检验了RNA从肾小球足细胞到肾小管上皮细胞的假说。我们开发了一种使用SLAMseq（用于组织中RNA代谢测序的SH连接烷基化）来追踪肾脏中exRNA的方法。我们将podocin-Cre小鼠与停止生长的UPRT小鼠杂交，以在足细胞中表达重组尿嘧啶磷酸核糖转移酶（UPRT）。给小鼠注射修饰的核碱基4-硫尿嘧啶，其仅在表达UPRT的细胞中以高效率掺入新生RNA中。我们收获肾小球或肾小管细胞，提取RNA并制备用于SLAMseq的文库，其中用4-硫尿嘧啶标记的mRNA位点在3 'UTR中检测为T>C转换。在肾小球中，我们检测到已知足细胞基因的标记，但不限于内皮细胞、肾小管或白色血细胞的基因。将错误发现率设定为1%，在4 TU处理的足细胞-UPRT小鼠中，被认为标记具有高置信度的基因比例为7.1%（95%置信区间6.8 - 7.4%），在Cre阴性对照中为2.5%（2.3 - 2.7%），在4 TU未处理对照中为1.0%（0.9 - 1.1%）。在肾小管细胞中，我们检测到与Cre阴性对照组相比，足细胞-UPRT小鼠中RNA标记的小幅但统计学显著性增加（零膨胀泊松回归模型中p = 7.4 × 10−16）。我们的结论是，RNA从足细胞转移到肾小管上皮细胞在体内生理条件下。我们的模型提供了探索这种新的信号通路在健康和肾脏疾病中的后果的机会。
There is a wealth of indirect evidence that extracellular RNA (exRNA) signalling can regulate renal tubular epithelial cell function. However, the physiological importance of this signalling is uncertain. We sought to determine the extent of extracellular RNA transfer between cells in a healthy kidney. We tested the hypothesis that RNA travels from glomerular podocytes to renal tubular epithelial cells. We developed a method to track exRNA in the kidney using SLAMseq (SH-linked alkylation for the metabolic sequencing of RNA in tissue). We crossed podocin-Cre mice with floxed-stop-UPRT mice to express recombinant uracil phosphoribosyl transferase (UPRT) in podocytes. Mice were injected with the modified nucleobase 4-thiouracil, which is incorporated into nascent RNA with high efficiency only in UPRT-expressing cells. We harvested glomeruli or tubular cells, extracted RNA and prepared libraries for SLAMseq, in which sites of mRNA labelling with 4-thiouracil are detected as T>C conversions in 3’UTRs. In glomeruli, we detected labelling of known podocyte genes but not of genes known to be restricted to endothelial, renal tubular or white blood cells. Setting a false-discovery rate of 1%, the proportion of genes deemed to be labelled with high confidence was 7.1% (95% confidence interval 6.8 – 7.4%) in 4TU-treated podocyte-UPRT mice, 2.5% (2.3 – 2.7%) in Cre-negative controls and 1.0% (0.9 – 1.1%) in 4TU-naïve controls. In tubular cells, we detected a small but statistically significant increase in RNA labelling in podocyte-UPRT mice compared to Cre-negative controls (p = 7.4 × 10−16 in a zero-inflated Poisson regression model). We conclude that RNA is transferred from podocytes to renal tubular epithelial cells in vivo under physiological conditions. Our model provides the opportunity to explore the consequences of this novel signalling pathway in health and kidney disease.