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

ABSTRACT: 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.

SUBMITTER: Miranda KC

PROVIDER: S-EPMC4451567 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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

Miranda Kevin C KC Bond Daniel T DT McKee Mary M Skog Johan J Păunescu Teodor G TG Da Silva Nicolas N Brown Dennis D Russo Leileata M LM

Kidney international 20100428 2

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 ...[more]

PMID: 20428099

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Project description:Biomarkers are of tremendous importance for the prediction, diagnosis, and observation of the therapeutic success of common complex multifactorial metabolic diseases, such as type II diabetes and obesity. However, the predictive power of the traditional biomarkers used (eg, plasma metabolites and cytokines, body parameters) is apparently not sufficient for reliable monitoring of stage-dependent pathogenesis starting with the healthy state via its initiation and development to the established disease and further progression to late clinical outcomes. Moreover, the elucidation of putative considerable differences in the underlying pathogenetic pathways (eg, related to cellular/tissue origin, epigenetic and environmental effects) within the patient population and, consequently, the differentiation between individual options for disease prevention and therapy - hallmarks of personalized medicine - plays only a minor role in the traditional biomarker concept of metabolic diseases. In contrast, multidimensional and interdependent patterns of genetic, epigenetic, and phenotypic markers presumably will add a novel quality to predictive values, provided they can be followed routinely along the complete individual disease pathway with sufficient precision. These requirements may be fulfilled by small membrane vesicles, which are so-called exosomes and microvesicles (EMVs) that are released via two distinct molecular mechanisms from a wide variety of tissue and blood cells into the circulation in response to normal and stress/pathogenic conditions and are equipped with a multitude of transmembrane, soluble and glycosylphosphatidylinositol-anchored proteins, mRNAs, and microRNAs. Based on the currently available data, EMVs seem to reflect the diverse functional and dysfunctional states of the releasing cells and tissues along the complete individual pathogenetic pathways underlying metabolic diseases. A critical step in further validation of EMVs as biomarkers will rely on the identification of unequivocal correlations between critical disease states and specific EMV signatures, which in future may be determined in rapid and convenient fashion using nanoparticle-driven biosensors.

Project description:BackgroundChronic renal disease (CKD) is a common and irreversible loss of renal function. Renal fibrosis reflected the degree of renal dysfunction. However, the current biomarkers only characterize the renal function instead of indicating the fibrosis degree. The potential diagnostic value of urinary exosomes derived circRNAs for renal fibrosis needs to be further studied.MethodsUrine exosomes from 3 chronic kidney disease (CKD) patients without renal fibrosis and 3 renal fibrotic patients were collected and human circRNAs microarray analysis were performed to detect the circRNAs expression profile. 110 biopsy-proven CKD patients and 54 healthy controls were enrolled and urine exosomes derived RNA was isolated. The expression of hsa_circ_0036649 was measured and the correlation with renal function parameter and pathological indicators was performed. The receiver operating characteristic (ROC) curve for the diagnosis of renal fibrosis was calculated.ResultsHuman circRNAs microarray showed 365 circRNAs up expressed and 195 circRNAs down expressed in renal fibrotic patients compared to none fibrosis CKD patients. The expression of hsa_circ_0036649 was decreased in renal fibrotic patients according to RT-PCR and correlated with serum creatinine, blood urea nitrogen (BUN), estimated glomerular filtration rate and cystatin c. Further, the expression of hsa_circ_0036649 was correlated with the score of tubulointerstitial fibrosis (TIF) and the score of glomerular sclerosis. The ROC curve showed that hsa_circ_0036649 may predict renal fibrosis at a cut-off value of 0.597 with a sensitivity of 45.5% and specificity of 87.9%.ConclusionExpression of urinary exosomes derived hsa_circ_0036649 associated with the degree of renal fibrosis. Its potential role as a biomarker in CKD remained to be supported by further follow-up studies.Key MessagescircRNAs profile in urine exosomes in renal fibrosis patients was revealed.The expression of urine exosomes derived hsa_circ_0036649 was correlated to renal function and fibrosis degree.circRNAs derived from urinary exosomes may become a new research direction for biomarkers of renal fibrosis.

Project description:Background and objectivesMicrovesicles and exosomes are involved in the pathogenesis of autosomal dominant polycystic kidney disease. However, it is unclear whether they also contribute to medullary sponge kidney, a sporadic kidney malformation featuring cysts, nephrocalcinosis, and recurrent kidney stones. We addressed this knowledge gap by comparative proteomic analysis.Design, setting, participants, & measurementsThe protein content of microvesicles and exosomes isolated from the urine of 15 patients with medullary sponge kidney and 15 patients with autosomal dominant polycystic kidney disease was determined by mass spectrometry followed by weighted gene coexpression network analysis, support vector machine learning, and partial least squares discriminant analysis to compare the profiles and select the most discriminative proteins. The proteomic data were verified by ELISA.ResultsA total of 2950 proteins were isolated from microvesicles and exosomes, including 1579 (54%) identified in all samples but only 178 (6%) and 88 (3%) specific for medullary sponge kidney microvesicles and exosomes, and 183 (6%) and 98 (3%) specific for autosomal dominant polycystic kidney disease microvesicles and exosomes, respectively. The weighted gene coexpression network analysis revealed ten modules comprising proteins with similar expression profiles. Support vector machine learning and partial least squares discriminant analysis identified 34 proteins that were highly discriminative between the diseases. Among these, CD133 was upregulated in exosomes from autosomal dominant polycystic kidney disease and validated by ELISA.ConclusionsOur data indicate a different proteomic profile of urinary microvesicles and exosomes in patients with medullary sponge kidney compared with patients with autosomal dominant polycystic kidney disease. The urine proteomic profile of patients with autosomal dominant polycystic kidney disease was enriched of proteins involved in cell proliferation and matrix remodeling. Instead, proteins identified in patients with medullary sponge kidney were associated with parenchymal calcium deposition/nephrolithiasis and systemic metabolic derangements associated with stones formation and bone mineralization defects.PodcastThis article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2019_04_24_CJASNPodcast_19_06_.mp3.

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

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

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