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Browsing by Subject "Single nuclear RNA sequencing"
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Item Alterations in Protein Translation and Carboxylic Acid Catabolic Processes in Diabetic Kidney Disease(MDPI, 2022-03-30) Collins, Kimberly S.; Eadon, Michael T.; Cheng, Ying-Hua; Barwinska, Daria; Ferreira, Ricardo Melo; McCarthy, Thomas W.; Janosevic, Danielle; Syed, Farooq; Maier, Bernhard; El-Achkar, Tarek M.; Kelly, Katherine J.; Phillips, Carrie L.; Hato, Takashi; Sutton, Timothy A.; Dagher, Pierre C.; Medicine, School of MedicineDiabetic kidney disease (DKD) remains the leading cause of end-stage kidney disease despite decades of study. Alterations in the glomerulus and kidney tubules both contribute to the pathogenesis of DKD although the majority of investigative efforts have focused on the glomerulus. We sought to examine the differential expression signature of human DKD in the glomerulus and proximal tubule and corroborate our findings in the db/db mouse model of diabetes. A transcriptogram network analysis of RNAseq data from laser microdissected (LMD) human glomerulus and proximal tubule of DKD and reference nephrectomy samples revealed enriched pathways including rhodopsin-like receptors, olfactory signaling, and ribosome (protein translation) in the proximal tubule of human DKD biopsy samples. The translation pathway was also enriched in the glomerulus. Increased translation in diabetic kidneys was validated using polyribosomal profiling in the db/db mouse model of diabetes. Using single nuclear RNA sequencing (snRNAseq) of kidneys from db/db mice, we prioritized additional pathways identified in human DKD. The top overlapping pathway identified in the murine snRNAseq proximal tubule clusters and the human LMD proximal tubule compartment was carboxylic acid catabolism. Using ultra-performance liquid chromatography-mass spectrometry, the fatty acid catabolism pathway was also found to be dysregulated in the db/db mouse model. The Acetyl-CoA metabolite was down-regulated in db/db mice, aligning with the human differential expression of the genes ACOX1 and ACACB. In summary, our findings demonstrate that proximal tubular alterations in protein translation and carboxylic acid catabolism are key features in both human and murine DKD.Item Deconvolution Tactics and Normalization in Renal Spatial Transcriptomics(Frontiers Media, 2022-01-13) Ferreira, Ricardo Melo; Freije, Benjamin J.; Eadon, Michael T.; Medicine, School of MedicineThe kidney is composed of heterogeneous groups of epithelial, endothelial, immune, and stromal cells, all in close anatomic proximity. Spatial transcriptomic technologies allow the interrogation of in situ expression signatures in health and disease, overlaid upon a histologic image. However, some spatial gene expression platforms have not yet reached single-cell resolution. As such, deconvolution of spatial transcriptomic spots is important to understand the proportion of cell signature arising from these varied cell types in each spot. This article reviews the various deconvolution strategies discussed in the 2021 Indiana O’Brien Center for Microscopy workshop. The unique features of Seurat transfer score methodology, SPOTlight, Robust Cell Type Decomposition, and BayesSpace are reviewed. The application of normalization and batch effect correction across spatial transcriptomic samples is also discussed.Item Identification of Novel Gene Regulatory Networks for Dystrophin Protein in Vascular Smooth Muscle Cells by Single-Nuclear Transcriptome Analysis(MDPI, 2023-03-14) Shen, Yan; Kim, Il-man; Tang, Yaoliang; Anatomy, Cell Biology and Physiology, School of MedicineDuchenne muscular dystrophy is an X-linked recessive disease caused by mutations in dystrophin proteins that lead to heart failure and respiratory failure. Dystrophin (DMD) is not only expressed in cardiomyocytes and skeletal muscle cells, but also in vascular smooth muscle cells (VSMCs). Patients with DMD have been reported to have hypotension. Single nuclear RNA sequencing (snRNA-seq) is a state-of-the-art technology capable of identifying niche-specific gene programs of tissue-specific cell subpopulations. To determine whether DMD mutation alters blood pressure, we compared systolic, diastolic, and mean blood pressure levels in mdx mice (a mouse model of DMD carrying a nonsense mutation in DMD gene) and the wide-type control mice. We found that mdx mice showed significantly lower systolic, diastolic, and mean blood pressure than control mice. To understand how DMD mutation changes gene expression profiles from VSMCs, we analyzed an snRNA-seq dataset from the muscle nucleus of DMD mutant (DMDmut) mice and control (Ctrl) mice. Gene Ontology (GO) enrichment analysis revealed that the most significantly activated pathways in DMDmut-VSMCs are involved in ion channel function (potassium channel activity, cation channel complex, and cation channel activity). Notably, we discovered that the DMDmut-VSMCs showed significantly upregulated expression of KCNQ5 and RYR2, whereas the most suppressed pathways were transmembrane transporter activity (such as anion transmembrane transporter activity, inorganic anion transmembrane transporter activity, import into cell, and import across plasma membrane). Moreover, we analyzed metabolic pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) using "scMetabolism" R package. DMDmut-VSMCs exhibited dysregulation of pyruvate metabolism and nuclear acid metabolism. In conclusion, via the application of snRNA-seq, we (for the first time) identify the potential molecular regulation by DMD in the upregulation of the expression of KCNQ5 genes in VSMCs, which helps us to understand the mechanism of hypotension in DMD patients. Our study potentially offers new possibilities for therapeutic interventions in systemic hypotension in DMD patients with pharmacological inhibition of KCNQ5.