Institute of Nephrology, Zhong Da Hospital, Southeast University School of Medicine, No. 87 Dingjiaqiao Rd, Nanjing, Jiangsu, China.
Wuxi People's Hospital Affiliated To Nanjing Medical University, Wuxi, Jiangsu, China.
J Transl Med. 2019 Feb 28;17(1):59. doi: 10.1186/s12967-019-1818-2.
Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease (ESKD) in the world. Emerging evidence has shown that urinary mRNAs may serve as early diagnostic and prognostic biomarkers of DKD. In this article, we aimed to first establish a novel bioinformatics-based methodology for analyzing the "urinary kidney-specific mRNAs" and verify their potential clinical utility in DKD.
To select candidate mRNAs, a total of 127 Affymetrix microarray datasets of diabetic kidney tissues and other tissues from humans were compiled and analyzed using an integrative bioinformatics approach. Then, the urinary expression of candidate mRNAs in stage 1 study (n = 82) was verified, and the one with best performance moved on to stage 2 study (n = 80) for validation. To avoid potential detection bias, a one-step Taqman PCR assay was developed for quantification of the interested mRNA in stage 2 study. Lastly, the in situ expression of the selected mRNA was further confirmed using fluorescent in situ hybridization (FISH) assay and bioinformatics analysis.
Our bioinformatics analysis identified sixteen mRNAs as candidates, of which urinary BBOX1 (uBBOX1) levels were significantly upregulated in the urine of patients with DKD. The expression of uBBOX1 was also increased in normoalbuminuric diabetes subjects, while remained unchanged in patients with urinary tract infection or bladder cancer. Besides, uBBOX1 levels correlated with glycemic control, albuminuria and urinary tubular injury marker levels. Similar results were obtained in stage 2 study. FISH assay further demonstrated that BBOX1 mRNA was predominantly located in renal tubular epithelial cells, while its expression in podocytes and urothelium was weak. Further bioinformatics analysis also suggested that tubular BBOX1 mRNA expression was quite stable in various types of kidney diseases.
Our study provided a novel methodology to identify and analyze urinary kidney-specific mRNAs. uBBOX1 might serve as a promising biomarker of DKD. The performance of the selected urinary mRNAs in monitoring disease progression needs further validation.
糖尿病肾病（DKD）是全球终末期肾病（ESKD）的主要病因。新出现的证据表明，尿 mRNA 可能作为 DKD 的早期诊断和预后生物标志物。在本文中，我们旨在首先建立一种新的基于生物信息学的方法来分析“尿肾脏特异性 mRNA”，并验证其在 DKD 中的潜在临床应用。
为了选择候选 mRNA，我们共编译并分析了 127 个人类糖尿病肾脏组织和其他组织的 Affymetrix 微阵列数据集，采用综合生物信息学方法。然后，在 1 期研究（n=82）中验证候选 mRNA 的尿表达，并将表现最佳的一个移至 2 期研究（n=80）进行验证。为了避免潜在的检测偏差，在 2 期研究中开发了一步 Taqman PCR 测定法来定量感兴趣的 mRNA。最后，使用荧光原位杂交（FISH）测定法和生物信息学分析进一步证实所选 mRNA 的原位表达。
我们的生物信息学分析确定了 16 个 mRNA 作为候选者，其中尿 BBOX1（uBBOX1）水平在 DKD 患者的尿液中显着上调。在正常白蛋白尿糖尿病患者中，uBBOX1 的表达也增加，而在尿路感染或膀胱癌患者中则保持不变。此外，uBBOX1 水平与血糖控制、蛋白尿和尿肾小管损伤标志物水平相关。在 2 期研究中也得到了类似的结果。FISH 测定法进一步表明，BBOX1 mRNA 主要位于肾小管上皮细胞中，而在足细胞和尿路上皮中的表达较弱。进一步的生物信息学分析还表明，各种类型的肾脏疾病中肾小管 BBOX1 mRNA 的表达相当稳定。
我们的研究提供了一种新的方法来识别和分析尿肾脏特异性 mRNA。uBBOX1 可能作为 DKD 的有前途的生物标志物。所选尿 mRNA 在监测疾病进展中的性能需要进一步验证。
