Identification of urinary exosomal noncoding RNAs as novel biomarkers in chronic kidney disease

Abstract

In chronic kidney disease (CKD), the decline in the glomerular filtration rate is associated with increased morbidity and mortality and thus poses a major challenge for healthcare systems. While the contribution of tissue-derived miRNAs and mRNAs to CKD progression has been extensively studied, little is known about the role of urinary exosomes and their association with CKD. Exosomes are small, membrane-derived endocytic vesicles that contribute to cell-to-cell communication and are present in various body fluids, such as blood or urine. Next-generation sequencing approaches have revealed that exosomes are enriched in noncoding RNAs and thus exhibit great potential for sensitive nucleic acid biomarkers in various human diseases. Therefore, in this study we aimed to identify urinary exosomal ncRNAs as novel biomarkers for diagnosis of CKD. Since up to now most approaches have focused on the class of miRNAs, we extended our analysis to several other noncoding RNA classes, such as tRNAs, tRNA fragments (tRFs), mitochondrial tRNAs, or lincRNAs. For their computational identification from RNA-seq data, we developed a novel computational pipeline, designated as ncRNASeqScan. By these analyses, in CKD patients we identified 30 differentially expressed ncRNAs, derived from urinary exosomes, as suitable biomarkers for early diagnosis. Thereby, miRNA-181a appeared as the most robust and stable potential biomarker, being significantly decreased by about 200-fold in exosomes of CKD patients compared to healthy controls. Using a cell culture system for CKD indicated that urinary exosomes might indeed originate from renal proximal tubular epithelial cells.

Keywords: chronic kidney disease; lincRNAs; miRNAs; ncRNAs; sequencing; tRFs; tRNAs; urinary exosomes.

FIGURE 1.

Classification of CKD. (A) The table represents the five stages of chronic kidney disease as assessed by analysis of the glomerular filtration rate (GFR). (B) The histogram displays the age distribution of healthy controls and CKD stages I–IV, including the number of females and males.

FIGURE 2.

Mapped noncoding RNA identified by RNA-seq. (A) Percentage of total number of reads mapped to noncoding RNAs. (B) Number of mapped unique exRNAs from different RNA species. (C) PCA plot showing a clear separation between CKD patients and healthy controls (HC). Each dot represents a sample, with different colors depicting the biological group to which each sample belongs.

FIGURE 3.

Differential abundance of ncRNA in exosomes of CKD. (A) The table represents the abundance of significant differences in the abundance of exosomal ncRNAs in CKD stages (ST) I–IV versus healthy controls (HC). (B) Venn diagram depicting the 30 overlapping differentially abundant exosomal ncRNAs between stages I, II, and IV of CKD and healthy controls. (C) Heatmap representing the relative abundance of 30 exosomal ncRNAs from stages I (n = 3), II (n = 4), IV (n = 5), and HC (n = 10). The color key indicates the expression change from negative (red) to positive (green). Rows represent the cluster of high abundance (orange) and reduced abundance (pink) of exosomal ncRNAs in CKD versus healthy controls.

FIGURE 4.

Nuclear encoded tRFs (A) and mitochondrial encoded tRNAs or mt-tRNAs (B). (A) The line graph represents length and number of reads of fragmented tRNAs: tRNA^Leu and tRNA^Val. The graphs show the presence of fragments at the 5′ in all cases. (X-axis) Length of the mitochondrial tRNAs; (y-axis) normalized mean reads in both CKD (red) and health controls (blue). (B) The line graph represents length and number of reads in three mitochondria tRNAs (mt-tRNA^Cys, mt-tRNA^Glu, mt-tRNA^Pro). (X-axis) Length of respective mitochondrial tRNAs; (y-axis) normalized mean reads in both CKD samples (red) and healthy controls (blue). Note that unlike in A, an abundance of full length tRNAs is observed.

FIGURE 5.

Identification of tRFs by Northern blotting. (A) Renal proximal tubule (RPTEC/TERT1) cells were differentiated and either left unstimulated (healthy controls) or stimulated (CKD) using IL-1β (10 ng/mL), TGF-β1 (10 ng/mL), and OSM (10 ng/mL). Total RNA was extracted from the cells (C) and supernatant, i.e., exosomes (E). (B) The levels of full-length tRNA^Leu and its derived tRF were quantified by phosphor imaging. The percentage (%) of tRF^Leu was compared relative to the total amounts of tRNA^Leu (i.e., full-length and fragment tRNA^Leu). Bar graph represents the mean ± SD from two independent experiments.