MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor beta 1: a novel role of miR-382.

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 ß1 (TGFß1), a model of epithelial-mesenchymal transition important for the development of renal interstitial fibrosis. Treatment of human renal epithelial cells with TGFß1 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 TGFß1, attenuated TGFß1-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 TGFß1, including superoxide dismutase 2 (SOD2). Knockdown of miR-382 attenuated TGFß1-induced downregulation of SOD2. Overexpression of SOD2 ameliorated TGFß1-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 TGFß1.

MicroRNA-target pairs in the rat kidney identified by microRNA microarray, proteomic, and bioinformatic analysis.

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 approximately 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.

Differential expression of cardiac mitochondrial proteins.

Mitochondria were isolated from whole hearts of Dahl salt sensitive (SS) and chromosome 13 consomic control (SS.13BN/Mcwi) rats using a mechanical homogenization process followed by density centrifugation. The proteins present in the two mitochondria preparations were quantified; equal amounts of protein from each sample were taken and trypsinized in the presence of either 16O or 18O before pooling. Incorporation of one or two 18O atoms at the C-terminus of the peptide cleaved by trypsin allows the distinction between the two samples. The proteins were identified by automated MS/MS sequencing and relative amounts of each protein assessed by comparison of the intensities of the constituent peptides. Relative quantification was performed using the ZoomQuant (v1.24) software. Nine proteins were found to be differentially expressed. Electron transfer flavoprotein alpha (P13803, ETFA) protein expression was two-fold lower in the SS compared to the SS.13BN. This was confirmed by Western blot and 2-DE gel quantification. Potential functional implications of this differential expression include an impaired capacity of the heart to oxidize fatty acids in the SS strain compared to the control. Mathematical modeling of mitochondrial electron transport predicted that the observed change in ETFA expression may result in decreased activity of the electron transport chain.

Renal regional proteomes in young Dahl salt-sensitive rats.

We performed an extensive proteomic analysis of the Dahl model of salt-sensitive hypertension. The consomic SS-13(BN) rat, genetically similar to the Dahl salt-sensitive rat, while exhibiting a significant amelioration of salt-induced hypertension, was used as a control. Proteomic analysis, using differential in-gel electrophoresis and mass spectrometry techniques, was performed in the renal cortex and the renal medulla of 6-week-old SS and SS-13(BN) rats before significant differences in blood pressure were developed between the 2 strains of rat. Several dozen proteins were identified as differentially expressed between SS and SS-13(BN) rats fed the 0.4% NaCl diet or switched to the 4% NaCl diet for 3 days (n=4). The identified proteins were involved in cellular functions or structures including signal transduction, energy metabolism, and the cytoskeleton. The proteomic analysis and subsequent Western blotting indicated that heterogeneous nuclear ribonucleoprotein K in the renal medulla was upregulated by the 4% NaCl diet in SS-13(BN) rats but downregulated in SS rats. The level of angiotensinogen mRNA in the renal medulla was regulated in an opposite manner. Silencing of heterogeneous nuclear ribonucleoprotein K resulted in an upregulation of angiotensinogen in cultured human kidney cells. In summary, we identified significant differences in kidney regional proteomic profiles between SS and SS-13(BN) rats and demonstrated a potential role of heterogeneous nuclear ribonucleoprotein K in the regulation of angiotensinogen expression in the renal medulla.