MicroRNA Sequence Profiles of Human Kidney Allografts With or Without Tubulointerstitial Fibrosis

Background. MicroRNA (miRNA) alterations accompanying interstitial fibrosis and tubular atrophy (IFTA) in kidney allografts may point toward pathologic mechanisms. Small-RNA sequencing provides information on total miRNA abundance and specific miRNA expression and allows analysis of differential expression based on read counts. Methods. MiRNA expression profiles of 8 human kidney allograft biopsies (4 IFTA and 4 normal biopsies, discovery set) were characterized using barcoded deep-sequencing of a cDNA library prepared from multiplexed RNA. Statistical analysis of the sequence data guided selection of miRNAs for validation and the levels of selected miRNAs were quantified in 18 biopsies (10 IFTA and 8 normal) using real-time quantitative PCR assays (RT-QPCR). Results. Total miRNA content was 50% lower in RNA from IFTA compared with normal biopsies. Global miRNA profiles clustered in partial agreement with diagnosis. Several miRNAs, including miR-21, 142-3p, and 5p and the cluster comprising miR-506 on chromosome X had twofold to sevenfold higher expression in IFTA compared with normal biopsies, whereas miRNA miR-30b and 30c were lower in IFTA biopsies (miR-30a, -30d, -30e, and all respective star sequences showed similar trends). IFTA and normal biopsy distinction was also noted in the pattern of miRNA nucleotide sequence variations. Differentially expressed miRNAs were confirmed on the larger set of allograft biopsies using RT-QPCR, and the levels of miRNAs were found to be associated with allograft function and survival. Conclusion. Differentially expressed miRNAs and their predicted targets identified by deep sequencing are candidates for further investigation to decipher the mechanism and management of kidney allograft fibrosis.