Abstract: TH-PO554
Single Glomerular Proteomics of Crescentic and Noncrescentic Glomeruli
Session Information
- Glomerular Diseases: Omics, Biomarkers, and Tools
October 24, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Authors
- Merchant, Michael, University of Louisville School of Medicine, Louisville, Kentucky, United States
- Biederman, Laura, The Ohio State University Wexner Medical Center, Columbus, Ohio, United States
- Barati, Michelle T., University of Louisville School of Medicine, Louisville, Kentucky, United States
- Gaut, Joseph, Washington University in St Louis School of Medicine, St Louis, Missouri, United States
- Dougherty, Julie, Nationwide Children's Hospital, Columbus, Ohio, United States
- Wilkey, Daniel Wade, University of Louisville School of Medicine, Louisville, Kentucky, United States
- Gaweda, Adam E., University of Louisville School of Medicine, Louisville, Kentucky, United States
- Klein, Jon B., University of Louisville School of Medicine, Louisville, Kentucky, United States
- Smoyer, William E., Nationwide Children's Hospital, Columbus, Ohio, United States
Background
Glomerular crescent formation is characteristic of rapidly progressive glomerulonephritis (RPGN), but the molecular mechanisms of crescent formation are poorly understood. We addressed the hypothesis that proteomic analysis of single crescentic glomeruli compared to unaffected glomerular tissue could reveal differential protein expression and provide mechanistic insight into crescent formation.
Methods
VIPER, a digital imaging platform, was used to annotate and select glomeruli with and without crescents in three different patient biopsies. Single glomeruli were followed through 5µm serial sections on PEN slides for microdissection. Isolated glomerular sections for crescentic and otherwise normal glomeruli (n=3) were analyzed using data independent acquisition (DIA) with a Thermo QE-HF LCMS. DIA data analysis (PeaksX) employed human glomerular and in silico spectral libraries. Single glomerulus datasets (n=6; 3x2) were quantitatively compared by paired and 2-way ttest (unadjusted p<0.05) and functionally characterized by g:Profiler using all proteins to establish differences within (paired) and between (2-way) crescentic and otherwise normal glomeruli.
Results
1,468 protein groups were detected across all datasets. 21 proteins differentiated crescentic and normal glomeruli with 19 more abundant in crescentic glomeruli. Ontologic analysis of all proteins identified top qualitative terms including cell adhesion (MF), cytoplasmic translation (BP), extracellular space (CC), coronavirus disease (KEGG), and primary focal segmental glomerulosclerosis (Wikipathways enrichment based on the following geneset: CR1, PTPRO, NPHS2, NPHS1, ILK, FYN, MYO1E, PODXL, LIMS1, CD151, MME, ITGA3, SYNPO, TLN1, ITGB1, AGRN, PARVA, DAG1, MYH9, ACTN4,I NF2, COL4A3, LAMA5, UTRN,C OL4A5, VCL, COL4A4, VTN, LAMB2, CD2AP, YWHAQ, PTK2, ITGAV, ITGB3, SCARB2, VIM, KRT8, CTNNB1).
Conclusion
This study demonstrates the ability to get glomerulus specific proteomic data from both crescentic and normal glomeruli from the same biopsy. The proteomic data shows a clear difference between the crescentic glomeruli and the normal glomeruli. Wider application of this technique will allow us to compare different stages of crescent formation as well as crescents in different disease states.
Funding
- NIDDK Support