A human glomerular SAGE transcriptome database

Background: To facilitate in the identification of gene products important in regulating renal glomerular structure and function, we have produced an annotated transcriptome database for normal human glomeruli using the SAGE approach. Description: The database contains 22,907 unique SAGE tag sequences, with a total tag count of 48,905. For each SAGE tag, the ratio of its frequency in glomeruli relative to that in 115 non-glomerular tissues or cells, a measure of transcript enrichment in glomeruli, was calculated. A total of 133 SAGE tags representing well-characterized transcripts were enriched 10-fold or more in glomeruli compared to other tissues. Comparison of data from this study with a previous human glomerular Sau3A-anchored SAGE library reveals that 47 of the highly enriched transcripts are common to both libraries. Among these are the SAGE tags representing many podocyte-predominant transcripts like WT-1, podocin and synaptopodin. Enrichment of podocyte transcript tags SAGE library indicates that other SAGE tags observed at much higher frequencies in this glomerular compared to non-glomerular SAGE libraries are likely to be glomerulus-predominant. A higher level of mRNA expression for 19 transcripts represented by glomerulus-enriched SAGE tags was verified by RT-PCR comparing glomeruli to lung, liver and spleen. Conclusions: The database can be retrieved from, or interrogated online at http://cgap.nci.nih.gov/SAGE. The annotated database is also provided as an additional file with gene identification for 9,022, and matches to the human genome or transcript homologs in other species for 1,433 tags. It should be a useful tool for in silico mining of glomerular gene expression

Massive-Scale RNA-Seq Analysis of Non Ribosomal Transcriptome in Human Trisomy 21

Hybridization- and tag-based technologies have been successfully used in Down syndrome to identify genes involved in various aspects of the pathogenesis. However, these technologies suffer from several limits and drawbacks and, to date, information about rare, even though relevant, RNA species such as long and small non-coding RNAs, is completely missing. Indeed, none of published works has still described the whole transcriptional landscape of Down syndrome. Although the recent advances in high-throughput RNA sequencing have revealed the complexity of transcriptomes, most of them rely on polyA enrichment protocols, able to detect only a small fraction of total RNA content. On the opposite end, massive-scale RNA sequencing on rRNA-depleted samples allows the survey of the complete set of coding and non-coding RNA species, now emerging as novel contributors to pathogenic mechanisms. Hence, in this work we analysed for the first time the complete transcriptome of human trisomic endothelial progenitor cells to an unprecedented level of resolution and sensitivity by RNA-sequencing. Our analysis allowed us to detect differential expression of even low expressed genes crucial for the pathogenesis, to disclose novel regions of active transcription outside yet annotated loci, and to investigate a plethora of non-polyadenilated long as well as short non coding RNAs. Novel splice isoforms for a large subset of crucial genes, and novel extended untranslated regions for known genes—possibly novel miRNA targets or regulatory sites for gene transcription—were also identified in this study. Coupling the rRNA depletion of samples, followed by high-throughput RNA-sequencing, to the easy availability of these cells renders this approach very feasible for transcriptome studies, offering the possibility of investigating in-depth blood-related pathological features of Down syndrome, as well as other genetic disorders

Overexpression of a Short Sulfonylurea Splice Variant Increases Cardiac Glucose Uptake and Uncouples Mitochondria by Regulating ROMK Activity

The mitochondrial splice variant of the sulfonylurea receptor (SUR2A-55) is associated with protection from myocardial ischemia-reperfusion (IR) injury, increased mitochondrial ATP sensitive K+ channel activity (mitoKATP) and altered glucose metabolism. While mitoKATP channels composed of CCDC51 and ABCB8 exist, the mitochondrial K+ pore regulated by SUR2A-55 is unknown. We explored whether SUR2A-55 regulates ROMK to form an alternate mitoKATP. We assessed glucose uptake in mice overexpressing SUR2A-55 (TGSUR2A−55) compared with WT mice during IR injury. We then examined the expression level of ROMK and the effect of ROMK modulation on mitochondrial membrane potential (Δψm) in WT and TGSUR2A−55 mice. TGSUR2A−55 had increased glucose uptake compared to WT mice during IR injury. The expression of ROMK was similar in WT compared to TGSUR2A−55 mice. ROMK inhibition hyperpolarized resting cardiomyocyte Δψm from TGSUR2A−55 mice but not from WT mice. In addition, TGSUR2A−55 and ROMK inhibitor treated WT isolated cardiomyocytes had enhanced mitochondrial uncoupling. ROMK inhibition blocked diazoxide induced Δψm depolarization and prevented preservation of Δψm from FCCP perfusion in WT and to a lesser degree TGSUR2A−55 mice. In conclusion, cardio-protection from SUR2A-55 is associated with ROMK regulation, enhanced mitochondrial uncoupling and increased glucose uptake

Accelerated lysine metabolism conveys kidney protection in salt-sensitive hypertension

Kidney metabolism in disease is important but not well understood. Here, using isotope-guided metabolomics, the authors show that lysine's metabolic activity conveys kidney protection in hypertension through accelerated metabolism and physiological effects on tubular function. Hypertension and kidney disease have been repeatedly associated with genomic variants and alterations of lysine metabolism. Here, we combined stable isotope labeling with untargeted metabolomics to investigate lysine's metabolic fate in vivo. Dietary C-13(6) labeled lysine was tracked to lysine metabolites across various organs. Globally, lysine reacts rapidly with molecules of the central carbon metabolism, but incorporates slowly into proteins and acylcarnitines. Lysine metabolism is accelerated in a rat model of hypertension and kidney damage, chiefly through N-alpha-mediated degradation. Lysine administration diminished development of hypertension and kidney injury. Protective mechanisms include diuresis, further acceleration of lysine conjugate formation, and inhibition of tubular albumin uptake. Lysine also conjugates with malonyl-CoA to form a novel metabolite N epsilon-malonyl-lysine to deplete malonyl-CoA from fatty acid synthesis. Through conjugate formation and excretion as fructoselysine, saccharopine, and N epsilon-acetyllysine, lysine lead to depletion of central carbon metabolites from the organism and kidney. Consistently, lysine administration to patients at risk for hypertension and kidney disease inhibited tubular albumin uptake, increased lysine conjugate formation, and reduced tricarboxylic acid (TCA) cycle metabolites, compared to kidney-healthy volunteers. In conclusion, lysine isotope tracing mapped an accelerated metabolism in hypertension, and lysine administration could protect kidneys in hypertensive kidney disease