Urinary Exosomes Contain MicroRNAs Capable of Paracrine Modulation of Tubular Transporters in Kidney

Abstract

Exosomes derived from all nephron segments are present in human urine, where their functionality is incompletely understood. Most studies have focused on biomarker discovery rather than exosome function. Through sequencing we identified the miRNA repertoire of urinary exosomes from healthy volunteers; 276 mature miRNAs and 345 pre-miRNAs were identified (43%/7% of reads). Among the most abundant were members of the miR-10, miR-30 and let-7 families. Targets for the identified miRNAs were predicted using five different databases; genes encoding membrane transporters and their regulators were enriched, highlighting the possibility that these miRNAs could modulate key renal tubular functions in a paracrine manner. As proof of concept, cultured renal epithelial cells were exposed to urinary exosomes and cellular exosomal uptake was confirmed; thereafter, reduced levels of the potassium channel ROMK and kinases SGK1 and WNK1 were observed in a human collecting duct cell line, while SPAK was unaltered. In proximal tubular cells, mRNA levels of the amino acid transporter gene SLC38A2 were diminished and reflected in a significant decrement of its encoded protein SNAT2. Protein levels of the kinase SGK1 did not change. Thus we demonstrated a novel potential function for miRNA in urinary exosomes.Urinary exosomes are lipid membrane-bound nanovesicles released from intracellular multivesicular bodies (MVBs) 1,2 and derived from all cells in the urinary tract [3][4][5] . During the inward budding of endosomes that give origin to exosomes, proteins 2 , mRNAs 6 , microRNAs (miRNAs) 7 , noncoding RNA (ncRNA) 8 , transcription factors 9 and other biomolecules present in the cytosol can be incorporated. The lipid bi-layer of these nanovesicles provides the cargo with stable storage conditions and protects it from degradation by extracellular proteases and ribonucleases 10. Studies in other tissues have shown that once exosomes and other microvesicles are released into the extracellular environment, interactions with cells can occur by direct ligand-receptor signalling, by exosomal fusion to the target cell membrane and discharge of exosomal content directly into the cytoplasm, or via phagocytosis/macropinocytosis 11,12 . Exosomes are known to deliver biologic cargo not only to neighbouring cells but also long distance 13 . The majority of studies concerning urinary exosomes have focused on their potential as biomarkers of disease pathology and progression, including prostate and bladder cancers [14][15][16][17] , but their functional significance is now being addressed. Inter-cellular signalling by exosomes in cultured murine renal epithelial cells was demonstrated for the first time by Street et al. 18 , who suggested that collecting duct cell-derived exosomes can transfer the ability to express AQP2. Our previous studies revealed that urinary exosomes inhibit bacterial growth of both commensal and uropathogenic E. coli by inducing bacterial lysis 19 . Bruschi and colleagues demonstrated that urinary exosomes can consume ...

Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery

“…An outcome of these studies is the ability to discern differences in the omics composition of urinary extracellular vesicles, correlate observed differences to health and acute or chronic renal disease, and use these observations to develop testable hypotheses addressing disease-specific mechanisms 74,118,130,132,133,169,170 . Furthermore, the utilization in these studies of second morning voids and spot urine samples to yield reproducible data suggests that the future for urinary extracellular vesicle diagnostics is encouraging.…”

Making urinary extracellular vesicles a clinically tractable source of biomarkers for inherited tubulopathies using a small volume precipitation method: proof of concept

“…The precipitation method yielded a similar quantity of protein and uEVs (on a per mL urine basis) compared to ultracentrifugation (as per our previously published protocol [7]); protein contents were 0.7-10.8 µg (0.7-10.8 µg/mL urine) and 12.1-50.8 µg (0.2-0.8 µg/mL urine) in uEV preparations produced by precipitation and ultracentrifugation respectively (n = 3; P = 0.2). Total number of uEVs from these preparations were 6.2-28.0 billion and 1.0-27.8 billion particles/mL urine respectively (n = 3; P = 0.4).…”

Novel Aspects of Extracellular Vesicles in the Regulation of Renal Physiological and Pathophysiological Processes

“…The decrease in SLC38A2 mRNA levels suggests lower mRNA stability due to the presence of targeting miRNAs in the vesicles. Similarly, PMCA1 and ROMK protein expression were down-regulated by uEVs in human collecting duct (HCD) cells (Gracia et al, 2017). This report indicates a potential regulatory role of EVs also in calcium and potassium reabsorption.…”

“…Additionally, a comprehensive study using next-generation sequencing evaluated the diversity of miRNAs present in uEVs and their impact on the expression of renal transporters and channels (Gracia et al, 2017). Based on the 10 most abundant miRNAs found (miR-10b-5p, miR-10a-5p, miR-30a-5p, miR-26a-5p, miR-22-3p, miR-204-5p, miR-181a-5p, miR-27b-3p, miR-30d-5p, miR-192-5p), the renal outer medullary potassium channel (ROMK, Kir1.1), the plasma membrane calcium-transporting ATPase (PMCA1) and the amino acid transporter SNAT2 (SLC38A2) appeared as candidates to be regulated by these EVs.…”