Kevin C. Miranda

Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

Urinary exosomes or microvesicles are being studied intensively to identify potential new biomarkers for renal disease. We sought to identify whether these microvesicles contain nucleic acids. We isolated microvesicles from human urine in the same density range as that previously described for urinary exosomes and found them to have an RNA integrity profile similar to that of kidney tissue, including 18S and 28S rRNA. This profile was better preserved in urinary microvesicles compared with whole cells isolated from urine, suggesting that microvesicles may protect RNA during urine passage. We were able to detect mRNA in the human urinary microvesicles encoding proteins from all regions of the nephron and the collecting duct. Further, to provide a proof of principle, we found that microvesicles isolated from the urine of the V-ATPase B1 subunit knockout mice lacked mRNA of this subunit while containing a normal amount of the B2 subunit and aquaporin 2. The microvesicles were found to be contaminated with extraneous DNA potentially on their surface; therefore, we developed a rapid and reliable means to isolate nucleic acids from within urine microvesicles devoid of this extraneous contamination. Our study provides an experimental strategy for the routine isolation and use of urinary microvesicles as a novel and non-invasive source of nucleic acids to further renal disease biomarker discovery.

Massively parallel sequencing of human urinary exosome/microvesicle RNA reveals a predominance of non-coding RNA.

Intact RNA from exosomes/microvesicles (collectively referred to as microvesicles) has sparked much interest as potential biomarkers for the non-invasive analysis of disease. Here we use the Illumina Genome Analyzer to determine the comprehensive array of nucleic acid reads present in urinary microvesicles. Extraneous nucleic acids were digested using RNase and DNase treatment and the microvesicle inner nucleic acid cargo was analyzed with and without DNase digestion to examine both DNA and RNA sequences contained in microvesicles. Results revealed that a substantial proportion (∼87%) of reads aligned to ribosomal RNA. Of the non-ribosomal RNA sequences, ∼60% aligned to non-coding RNA and repeat sequences including LINE, SINE, satellite repeats, and RNA repeats (tRNA, snRNA, scRNA and srpRNA). The remaining ∼40% of non-ribosomal RNA reads aligned to protein coding genes and splice sites encompassing approximately 13,500 of the known 21,892 protein coding genes of the human genome. Analysis of protein coding genes specific to the renal and genitourinary tract revealed that complete segments of the renal nephron and collecting duct as well as genes indicative of the bladder and prostate could be identified. This study reveals that the entire genitourinary system may be mapped using microvesicle transcript analysis and that the majority of non-ribosomal RNA sequences contained in microvesicles is potentially functional non-coding RNA, which play an emerging role in cell regulation.

An Extended Nomenclature for Mammalian V-ATPase Subunit Genes and Splice Variants

The vacuolar-type H+-ATPase (V-ATPase) is a multisubunit proton pump that is involved in both intra- and extracellular acidification processes throughout the body. Multiple homologs and splice variants of V-ATPase subunits are thought to explain its varied spatial and temporal expression pattern in different cell types. Recently subunit nomenclature was standardized with a total of 22 subunit variants identified. However this standardization did not accommodate the existence of splice variants and is therefore incomplete. Thus, we propose here an extension of subunit nomenclature along with a literature and sequence database scan for additional V-ATPase subunits. An additional 17 variants were pulled from a literature search while 4 uncharacterized potential subunit variants were found in sequence databases. These findings have been integrated with the current V-ATPase knowledge base to create a new V-ATPase subunit catalogue. It is envisioned this catalogue will form a new platform on which future studies into tissue- and organelle-specific V-ATPase expression, localization and function can be based.