BackgroundExosomes, endosome-derived membrane microvesicles, contain specific RNA transcripts that are thought to be involved in cell-cell communication. These RNA transcripts have great potential as disease biomarkers. To characterize exosomal RNA profiles systemically, we performed RNA sequencing analysis using three human plasma samples and evaluated the efficacies of small RNA library preparation protocols from three manufacturers. In all we evaluated 14 libraries (7 replicates).ResultsFrom the 14 size-selected sequencing libraries, we obtained a total of 101.8 million raw single-end reads, an average of about 7.27 million reads per library. Sequence analysis showed that there was a diverse collection of the exosomal RNA species among which microRNAs (miRNAs) were the most abundant, making up over 42.32% of all raw reads and 76.20% of all mappable reads. At the current read depth, 593 miRNAs were detectable. The five most common miRNAs (miR-99a-5p, miR-128, miR-124-3p, miR-22-3p, and miR-99b-5p) collectively accounted for 48.99% of all mappable miRNA sequences. MiRNA target gene enrichment analysis suggested that the highly abundant miRNAs may play an important role in biological functions such as protein phosphorylation, RNA splicing, chromosomal abnormality, and angiogenesis. From the unknown RNA sequences, we predicted 185 potential miRNA candidates. Furthermore, we detected significant fractions of other RNA species including ribosomal RNA (9.16% of all mappable counts), long non-coding RNA (3.36%), piwi-interacting RNA (1.31%), transfer RNA (1.24%), small nuclear RNA (0.18%), and small nucleolar RNA (0.01%); fragments of coding sequence (1.36%), 5′ untranslated region (0.21%), and 3′ untranslated region (0.54%) were also present. In addition to the RNA composition of the libraries, we found that the three tested commercial kits generated a sufficient number of DNA fragments for sequencing but each had significant bias toward capturing specific RNAs.ConclusionsThis study demonstrated that a wide variety of RNA species are embedded in the circulating vesicles. To our knowledge, this is the first report that applied deep sequencing to discover and characterize profiles of plasma-derived exosomal RNAs. Further characterization of these extracellular RNAs in diverse human populations will provide reference profiles and open new doors for the development of blood-based biomarkers for human diseases.

The human miR-29 family of microRNAs has three mature members, miR-29a, miR-29b, and miR-29c. miR-29s are encoded by two gene clusters. Binding sites for several transcriptional factors have been identified in the promoter regions of miR-29 genes. The miR-29 family members share a common seed region sequence and are predicted to target largely overlapping sets of genes. However, the miR-29 family members exhibit differential regulation in several cases and different subcellular distribution, suggesting their functional relevance may not be identical. miR-29s directly target at least 16 extracellular matrix genes, providing a dramatic example of a single microRNA targeting a large group of functionally related genes. Strong antifibrotic effects of miR-29s have been demonstrated in heart, kidney, and other organs. miR-29s have also been shown to be proapoptotic and involved in the regulation of cell differentiation. It remains to be explored how various cellular effects of miR-29s determine functional relevance of miR-29s to specific diseases and how the miR-29 family members may function cooperatively or separately.

Abstract-Dahl salt-sensitive (SS) rats exhibit increased renal medullary oxidative stress and blood pressure salt-sensitivity compared with consomic, salt-resistant SS-13 BN rats, despite highly similar genetic backgrounds. The present study examined potential sources of renal medullary superoxide in prehypertensive SS rats fed a 0.4% NaCl diet by assessing activity and protein levels of superoxide producing and scavenging enzymes. Superoxide production was nearly doubled in SS rats compared with SS-13 BN rats as determined by urinary 8-isoprostane excretion and renal medullary oxy-ethidium microdialysate levels. Medullary superoxide production in tissue homogenates was greater in SS rats, and the NADPH oxidase inhibitor diphenylene iodonium preferentially reduced SS levels to those found in SS-13 BN rats. Dinitrophenol, a mitochondrial uncoupler, eliminated the remaining superoxide production in both strains, whereas inhibition of xanthine oxidase, NO synthase, and cycloxygenase had no effect. L-arginine, NO synthase, superoxide dismutase, catalase, and glutathione peroxidase activities between SS and SS-13 BN rats did not differ. Chronic blood pressure responses to a 4% NaCl diet were then determined in the presence or absence of the NADPH oxidase inhibitor apocynin (3.5 g/kg per minute), chronically delivered directly into the renal medulla. Apocynin infusion reduced renal medullary interstitial superoxide from 1059Ϯ130 to 422Ϯ80 (oxyethidium fluorescence units) and mean arterial pressure from 175Ϯ4 to 157Ϯ6 mm Hg in SS rats, whereas no effects on either were observed in the SS-13 BN . We conclude that excess renal medullary superoxide production in SS rats contributes to salt-induced hypertension, and NADPH oxidase is the major source of the excess superoxide.

Delayed ischemic preconditioning effectively protects kidneys from ischemia-reperfusion injury but the mechanism underlying renal protection remains poorly understood. Here we examined the in vivo role of microRNA miR-21 in the renal protection conferred by delayed ischemic preconditioning in mice. A 15 minute renal ischemic preconditioning significantly increased the expression of miR-21 by 4 hours and substantially attenuated ischemia-reperfusion injury induced 4 days later. A locked nucleic acid-modified anti-miR-21 given at the time of ischemic preconditioning knocked down miR-21 and significantly exacerbated subsequent ischemia-reperfusion injury in the mouse kidney. Knockdown of miR-21 resulted in significant upregulation of programmed cell death protein 4, a pro-apoptotic target gene of miR-21, and substantially increased tubular cell apoptosis. Hypoxia inducible factor-1α in the kidney was activated after ischemic preconditioning and blockade of its activity with a decoy abolished the up-regulation of miR-21 in cultured human renal epithelial cells treated with the inducer cobalt chloride. In the absence of ischemic preconditioning, knockdown of miR-21 alone did not significantly affect ischemia-reperfusion injury in the mouse kidney. Thus, upregulation of miR-21 contributes to the protective effect of delayed ischemic preconditioning against subsequent renal ischemia-reperfusion injury.

Mammalian genomes contain several hundred highly conserved genes encoding microRNAs. In silico analysis has predicted that a typical microRNA may regulate the expression of hundreds of target genes, suggesting miRNAs might have broad biological significance. A major challenge is to obtain experimental evidence for predicted microRNA–target pairs. We reasoned that reciprocal expression of a microRNA and a predicted target within a physiological context would support the presence and relevance of a microRNA–target pair. We used microRNA microarray and proteomic techniques to analyze the cortex and the medulla of rat kidneys. Of the 377 microRNAs analyzed, we identified 6 as enriched in the renal cortex and 11 in the renal medulla. From ∼2100 detectable protein spots in two-dimensional gels, we identified 58 proteins as more abundant in the renal cortex and 72 in the renal medulla. The differential expression of several microRNAs and proteins was verified by real-time PCR and Western blot analyses, respectively. Several pairs of reciprocally expressed microRNAs and proteins were predicted to be microRNA–target pairs by TargetScan, PicTar, or miRanda. Seven pairs were predicted by two algorithms and two pairs by all three algorithms. The identification of reciprocal expression of microRNAs and their computationally predicted targets in the rat kidney provides a unique molecular basis for further exploring the biological role of microRNA. In addition, this study establishes a differential profile of microRNA expression between the renal cortex and the renal medulla and greatly expands the known differential proteome profiles between the two kidney regions.

Abstract-MicroRNAs are endogenous repressors of gene expression. We examined microRNAs in the renal medulla of Dahl salt-sensitive rats and consomic SS-13 BN rats. Salt-induced hypertension and renal injury in Dahl salt-sensitive rats, particularly medullary interstitial fibrosis, have been shown previously to be substantially attenuated in SS-13 BN rats. Of 377 microRNAs examined, 5 were found to be differentially expressed between Dahl salt-sensitive rats and consomic SS-13 BN rats receiving a high-salt diet. Real-time PCR analysis demonstrated that high-salt diets induced substantial upregulation of miR-29b in the renal medulla of SS-13 BN rats but not in SS rats. miR-29b was predicted to regulate 20 collagen genes, matrix metalloproteinase 2 (Mmp2), integrin ␤1 (Itgb1), and other genes related to the extracellular matrix. Expression of 9 collagen genes and Mmp2 was upregulated by a high-salt diet in the renal medulla of SS rats, but not in SS-13 BN rats, an expression pattern opposite to miR-29b. Knockdown of miR-29b in the kidneys of SS-13 BN rats resulted in upregulation of several collagen genes. miR-29b reduced expression levels of several collagen genes and Itgb1 in cultured rat renal medullary epithelial cells. Moreover, miR-29b suppressed the activity of luciferase when the reporter gene was linked to a 3Ј-untranslated segment of collagen genes Col1a1, Col3a1, Col4a1, Col5a1, Col5a2, Col5a3, Col7a1, Col8a1, Mmp2, or Itgb1 but not Col12a1. The result demonstrated broad effects of miR-29b on a large number of collagens and genes related to the extracellular matrix and suggested involvement of miR-29b in the protection from renal medullary injury in SS-13 BN rats. (Hypertension. 2010;55:974-982.)

SUMMARY NAD(P)H oxidase has been shown to be important in the development of salt-sensitive hypertension. Here we show that the expression of a subunit of NAD(P)H oxidase, p67phox, was increased in response to a high salt diet in the outer renal medulla of the Dahl salt-sensitive (SS) rat, an animal model for human salt-sensitive hypertension. The higher expression of p67phox, not the other subunits observed, was associated with higher NAD(P)H oxidase activity and salt-sensitivity in SS rats compared with a salt-resistant strain. Genetic mutations of the SS allele of p67phox were found in the promoter region and contributed to higher promoter activity than that of the salt-resistant strain. To verify the importance of p67phox, we disrupted p67phox in SS rats using zinc finger nucleases technology. These rats exhibited a significant reduction of salt-sensitive hypertension and renal medullary oxidative stress and injury. p67phox could represent a target for salt-sensitive hypertension therapy.

We reported previously an approach for identifying microRNA (miRNA)-target pairs by combining miRNA and proteomic analyses. The approach was applied in the present study to examine human renal epithelial cells treated with transforming growth factor β1 (TGFβ1), a model of epithelial–mesenchymal transition important for the development of renal interstitial fibrosis. Treatment of human renal epithelial cells with TGFβ1 resulted in upregulation of 16 miRNAs and 18 proteins and downregulation of 17 miRNAs and 16 proteins. Of the miRNAs and proteins that exhibited reciprocal changes in expression, 77 pairs met the sequence criteria for miRNA–target interactions. Knockdown of miR-382, which was up-regulated by TGFβ1, attenuated TGFβ1-induced loss of the epithelial marker E-cadherin. miR-382 was confirmed by 3′-untranslated region reporter assay to target five genes that were downregulated at the protein level by TGFβ1, including superoxide dismutase 2 (SOD2). Knockdown of miR-382 attenuated TGFβ1-induced downregulation of SOD2. Overexpression of SOD2 ameliorated TGFβ1-induced loss of the epithelial marker. The study provided experimental evidence in the form of reciprocal expression at the protein level for a large number of predicted miRNA-target pairs and discovered a novel role of miR-382 and SOD2 in the loss of epithelial characteristics induced by TGFβ1.