Clinical utility of extracellular RNA as marker of kidney disease progression

描述（由申请人提供）： 慢性肾脏疾病（CKD）是导致心血管疾病、感染和癌症过早死亡的原因，是人类遭受巨大痛苦和社会成本的原因。大部分CKD患者发展为终末期肾病，需要透析或肾移植。识别有CKD进展风险的患者可能有助于精准用药并预防伴随的并发症。开发预测CKD进展的机制生物标志物可能有助于开发新的治疗方法。非编码RNA（ncRNA），特别是microRNA（miRNA）越来越多地被认为是通过调节基因表达影响多种细胞过程的生物分子。已知组织RNA表达传达关于疾病状态的信息。最近，已经认识到细胞外RNA（exRNA）通过其包围的微泡而被保护免受核酸酶的影响，因此可以在包括尿液的生物流体中测量，并传达病理生理学知识。我们的实验室在RNA表达的高通量分析方面拥有丰富的经验。我们已经开发了一种cDNA深度测序方法，以具有成本效益的，全转录组和偏倚降低的方式在多个样品中分析miRNA。通过研究数千种不同的人类样本，我们发现并策划了miRNA和其他小RNA。目前，我们正在开发一种平行的方法来筛选所有的ncRNA。我们还采用了定量逆转录聚合酶链反应（qRT-PCR）技术，以便在更大的天然和移植肾脏疾病患者研究队列中进行无创验证和确认。我们建议将我们的方法应用于从CKD患者收集的尿液标本中全面分析miRNA和其他ncRNA。我们的目标是鉴定尿液中可以预测CKD进展并开发预后生物标志物的exRNA。我们提出了一个2阶段的方案;发现阶段应用深度测序进行转录组范围的分析和候选标志物的鉴定，以及验证阶段应用RT-qPCR来表征额外的样品并鉴定发现的结果。为了快速实施我们的研究计划，我们与进行CKD纵向队列研究的研究者建立了合作关系。尿液标本与丰富的人口统计学、临床和肾脏结局数据相关，可从成人和儿童队列研究中获得;肾小球和非肾小球疾病;天然肾脏和肾移植受者。这允许发现能够预测整个肾脏疾病谱的进展的生物标志物，因为CKD进展的机制在很大程度上独立于CKD病因。尽管如此，我们的研究是针对亚组特异性生物标志物的发现。我们的初步研究结果证实了在生物流体中分析exRNA的可行性，并检测与临床相关结果的关联。我们预测，我们的研究结果将为基础科学家提供肾脏疾病进展中RNA失调的信息，并为临床试验提供临床科学家生物标志物候选人。

DESCRIPTION (provided by applicant): Chronic kidney disease (CKD) is responsible for premature death from cardiovascular disease, infections and cancer, considerable human suffering and social costs. A large proportion of CKD patients develop end stage renal disease and require dialysis or kidney transplantation. Identifying patients at risk for CKD progression may facilitate precision medicine and prevent attendant complications. Development of mechanistic biomarkers predictive of CKD progression may help develop new treatment(s). Non-coding RNA (ncRNA), specifically microRNA (miRNA) is increasingly recognized as biomolecules that affect multiple cellular processes via regulation of gene expression. It is known that tissue RNA expression conveys information on disease status. Recently, it has been recognized that extracellular RNA (exRNA) is protected from nucleases by their encompassing microvesicles, and therefore can be measured in biological fluids including urine, and convey pathophysiological knowledge. Our labs have extensive experience in high throughput analysis of RNA expression. We have developed a cDNA deep sequencing method to profile miRNA in multiple samples, in a cost-effective, transcriptome-wide and bias-reduced manner. By studying thousands of diverse human samples we have discovered and curated miRNA and other small RNA. Currently, we are developing a parallel method to screen for all ncRNA. We have also adapted quantitative reverse-transcription polymerase chain reaction (qRT-PCR) technology to allow non- invasive verification and validation in larger study cohorts of patients with native and transplant kidney disease. We propose to apply our methods to comprehensively profile miRNA and other ncRNA in urine specimens collected from CKD patients. We aim to identify exRNA in the urine that can predict progression of CKD and develop prognostic biomarkers. We propose a 2-stage protocol; a discovery stage applying deep sequencing for transcriptome-wide profiling and identification of candidate markers, and a validation phase applying RT- qPCR to characterize additional samples and qualify the discovered findings. To allow rapid implementation of our research plan we established collaborations with investigators conducting longitudinal cohort studies of CKD. Urine specimens, linked with rich demographic, clinical and kidney outcome data are available from studies of both adult and pediatric cohorts; both glomerular and non-glomerular diseases; in both native kidneys and kidney transplant recipients. This permits discovery of biomarkers capable of predicting progression across the spectrum of kidney diseases as mechanisms of CKD progression are to a largely independent of CKD etiology. Nonetheless, our studies are powered for subgroup-specific biomarker discovery. Our preliminary findings confirm feasibility to profile exRNA in biofluids, and to detect associations with clinical relevant outcomes. We predict that our findings will inform basic scientist on RNA dysregulation in kidney disease progression and provide clinical scientist biomarker candidates for clinical trials.