Bioinformatics-based discovery of the urinary BBOX1 mRNA as a potential biomarker of diabetic kidney disease.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.