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.

Recent advances in mass spectrometry have enabled system-wide analyses of protein turnover. By globally quantifying the kinetics of protein clearance and synthesis, these methodologies can provide important insights into the regulation of the proteome under varying cellular and environmental conditions. To facilitate such analyses, we have employed a methodology that combines metabolic isotopic labeling (Stable Isotope Labeling in Cell Culture - SILAC) with isobaric tagging (Tandem Mass Tags - TMT) for analysis of multiplexed samples. The fractional labeling of multiple time-points can be measured in a single mass spectrometry run, providing temporally resolved measurements of protein turnover kinetics. To demonstrate the feasibility of the approach, we simultaneously measured the kinetics of protein clearance and accumulation for more than 3000 proteins in dividing and quiescent human fibroblasts and verified the accuracy of the measurements by comparison to established non-multiplexed approaches. The results indicate that upon reaching quiescence, fibroblasts compensate for lack of cellular growth by globally downregulating protein synthesis and upregulating protein degradation. The described methodology significantly reduces the cost and complexity of temporally-resolved dynamic proteomic experiments and improves the precision of proteome-wide turnover data.

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.

Abstract-In a previous proteomic study, we found dramatic differences in fumarase in the kidney between Dahl salt-sensitive rats and salt-insensitive consomic SS-13 BN rats. Fumarase catalyzes the conversion between fumarate and L-malate in the tricarboxylic acid cycle. Little is known about the pathophysiological significance of fumarate metabolism in cardiovascular and renal functions, including salt-induced hypertension. The fumarase gene is located on the chromosome substituted in the SS-13 BN rat. Sequencing of fumarase cDNA indicated the presence of lysine at amino acid position 481 in Dahl salt-sensitive rats and glutamic acid in Brown Norway and SS-13 BN rats. Total fumarase activity was significantly lower in the kidneys of Dahl salt-sensitive rats compared with SS-13 BN rats, despite an apparent compensatory increase in fumarase abundance in Dahl salt-sensitive rats. Intravenous infusion of a fumarate precursor in SS-13 BN rats resulted in a fumarate excess in the renal medulla comparable to that seen in Dahl salt-sensitive rats. The infusion significantly exacerbated salt-induced hypertension in SS-13 BN rats (140Ϯ3 vs125Ϯ2 mm Hg in vehicle control at day 5 on a 4% NaCl diet; PϽ0.05). In addition, the fumarate infusion increased renal medullary tissue levels of H 2 O 2 . Treatment of cultured human renal epithelial cells with the fumarate precursor also increased cellular levels of H 2 O 2 . These data suggest a novel role for fumarate metabolism in salt-induced hypertension and renal medullary oxidative stress. BN rat has the same genomic makeup as the SS rat except for chromosome 13, which is introgressed from the Brown Norway (BN) rat and substantially attenuates salt-sensitive hypertension and renal injury. 3 A tree-like network of molecular, biochemical, and physiological mechanisms is likely involved in the development of Dahl salt-sensitive hypertension and renal injury. 4 Comparative analysis of SS and SS-13 BN rats has revealed several new components of this regulatory network. Examples include increased levels of superoxide and H 2 O 2 , 5,6 dysregulation of 11␤-hydroxysteroid dehydrogenase, and alterations of glucocorticoid metabolism 4 in the renal medulla of SS rats compared with SS-13 BN rats. Additional mechanisms and particularly sequence variations of specific genes involved in the SS phenotypes remain to be discovered or validated. 4,[7][8][9] Fumarase was one of the proteins exhibiting dramatic differences between SS and SS-13 BN rats according to a recent proteomic study. 10 The analysis indicated a consistent and substantial difference in the isoelectric point of fumarase in SS and SS-13 BN rats, as reflected by a significant shift of the protein spot on 2D gels. Fumarase catalyzes the reversible conversion between fumarate and L-malate in the tricarboxylic acid cycle in mitochondria. Rare loss-of-function mutations of fumarase in humans cause accumulation of fumarate and are associated with the development of hereditary leiomyomatosis, renal cell cancer, or encephalopathy. [11][1...

The awareness, treatment, and control rates of hypertension for young adults are much lower than average. It is urgently needed to explore the variances of metabolic profiles for early diagnosis and treatment of hypertension. In current study, we applied a GC–MS based metabolomics platform coupled with a network approach to analyze plasma samples from young hypertensive men and age-matched healthy controls. Our findings confirmed distinct metabolic footprints of young hypertensive men. The significantly altered metabolites between two groups were enriched for the biological module of amino acids biosynthesis. The correlations of GC–MS metabolomics data were then visualized as networks based on Pearson correlation coefficient (threshold = 0.6). The plasma metabolites identified by GC–MS and the significantly altered metabolites (P < 0.05) between patients and controls were respectively included as nodes of a network. Statistical and topological characteristics of the networks were studied in detail. A few amino acids, glycine, lysine, and cystine, were screened as hub metabolites with higher values of degree (k), and also obtained highest scores of three centrality indices. The short average path lengths and high clustering coefficients of the networks revealed a small-world property, indicating that variances of these amino acids have a major impact on the metabolic change in young hypertensive men. These results suggested that disorders of amino acid metabolism might play an important role in predisposing young men to developing hypertension. The combination of metabolomics and network methods would provide another perspective on expounding the molecular mechanism underlying complex diseases.

Abstract-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 (nϭ4). 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. (Hypertension. 2008;51:899-904.)

MicroRNA (miRNA) has emerged rapidly as a major new direction in many fields of research including kidney and blood pressure research. A mammalian genome encodes several hundred miRNAs. These miRNAs potentially regulate the expression of thousands of proteins. miRNA expression profiles differ substantially between the kidney and other organs as well as between kidney regions. miRNAs may be functionally important in models of diabetic nephropathy, podocyte development, and polycystic disease. miRNAs may be involved in the regulation of arterial blood pressure, including possible involvement in genetic elements of hypertension. Studies of miRNAs could generate diagnostic biomarkers for kidney disease and new mechanistic insights into the complex regulatory networks underlying kidney disease and hypertension. Further progress in the understanding of miRNA biogenesis and action and technical improvements for target identification and miRNA manipulation will be important for studying miRNAs in renal function and blood pressure regulation.

Fumarase catalyzes the interconversion of fumarate and L-malate in the tricarboxylic acid cycle. The Dahl salt-sensitive (SS) rat, a model of salt-sensitive hypertension, exhibits fumarase insufficiencies. To investigate the mechanism mediating the effect of fumarase-related metabolites on hypertension, we considered the pathway in which L-malate can be converted to oxaloacetate, aspartate, argininosuccinate, and L-arginine, the substrate of nitric oxide (NO) synthase. The levels of aspartate, citrulline, L-arginine, and NO were significantly decreased in the kidneys of SS rats compared to salt-insensitive consomic SS.13 rats. Knockdown of fumarase in human kidney cells and vascular endothelial cells resulted in decreased levels of malate, aspartate, L-arginine, and NO. Supplementation of aspartate or malate increased renal levels of L-arginine and NO and attenuated hypertension in SS rats. These findings reveal a multi-step metabolic pathway important for hypertension in which malate and aspartate may modulate blood pressure by altering levels of L-arginine and NO.