Background/Purpose: Lupus nephritis (LN) is a common manifestation of SLE associated with significant morbidity and mortality. microRNAs (miRs) are small non-coding RNAs that regulate translation and mRNA stability. Preliminary studies have reported changes in miR expression in kidney tissue, urine, and PBMC that correlated with disease activity in LN. However, use of RNA deep-sequencing methods has not been previously described. We aimed at identifying miR expression patterns in LN by RNA sequencing.

Methods: Cell-free urine supernatants from adult (n=9) and pediatric (n=4) female patients with LN were obtained at the time of active disease and during remission. Total RNA was used to prepare small RNA cDNA libraries for Illumina sequencing. Multiplexing through sample-specific 3′ adapters (“bar coding”) was applied to limit batch effects, labor and cost. Sequenced reads were mapped to the human genome and small RNA databases, and miRs were quantified by relative read abundance. qRT-PCR was used for quantitative validation of sequencing results.

Results: We were able to obtain reproducible profiles of miRNA from the small RNA fraction. In a paired-sample analysis comparing the number of miR sequence reads in urine of active versus inactive LN, we found significant upregulation of multiple miRNAs, including -185, -328, -378, -874, and -423. In total, we found differential expression of ~19 miRs during active nephritis. A subset analysis revealed 13 miRs that were upregulated by a 400-1,000 fold change during active disease in pediatric, but not in adult samples. Differential expression of several miRs was confirmed by miRNA qRT-PCR.

Conclusion: In summary, we detected a group of miRs (most of which have not been previously described in lupus) with significantly higher presence in the urine during active LN, particularly, in pediatric patients. These miRs may represent biomarkers for disease activity or indicators of specific histologic features. Several urine miRs were previously found to be differentially expressed in immune cells, which may imply that their presence in urine originates from infiltrating rather than kidney resident cells. Finally, upregulated miRs during active LN could imply that their “protective” target genes are repressed in relapse, and that identifying the latter may reveal novel therapeutic pathways in this challenging disease.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/next-generation-sequencing-of-urinary-microrna-in-human-lupus-nephritis/