Oxford Kidney Pathology Atlas: Single Cellular Profiling of Renal Biopsy Tissue
- Glomerular Diseases: From Inflammation to Fibrosis - III
November 04, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: From Inflammation to Fibrosis
- Kepple, Jessica, University of Oxford, Oxford, United Kingdom
- Davis, Simon, University of Oxford, Oxford, United Kingdom
- Fischer, Roman, University of Oxford, Oxford, United Kingdom
- Bull, Katherine R., University of Oxford, Oxford, United Kingdom
Group or Team Name
- Oxford Renal Pathology Research Group-Bull.
Glomerular damage is central to many kidney diseases, thus a better understanding of glomerular cell pathology is essential for developing more targeted therapeutics. Transcriptomic and proteomic profiling of human renal tissue may address this challenge. Many studies utilize nephrectomy sections, with limited samples, technical glomerular dissociation bias, and non-standardized methodology restricting the research application of biopsies. As part of the Oxford Kidney Pathology Atlas study, we developed experimental protocols for processing standard human renal biopsy samples for single-nuclei RNA sequencing (snRNA-Seq) and glomerular proteomics and generated pilot data from healthy donors and an early primary idiopathic nephrotic syndrome (iNS) case.
Biopsy cores from diseased patients and pre-transplantation controls were processed for clinical histology, snRNA-Seq and targeted glomerular proteomics. SnRNA-Seq libraries were analyzed using the R package Seurat. Glomerular regions from formalin-fixed paraffin-embedded (FFPE) renal biopsy sections were isolated via targeted laser capture microdissection coupled with mass spectrometry (MS). MS results were analyzed using DI-ANN and Perseus software to assess relative protein abundance.
We conducted a targeted proteomics titration experiment with varying numbers of isolated glomeruli from healthy donors to establish the minimum material required for proteomics. We isolated glomerular proteins from FFPE-embedded renal section, quantifying over 4.5k unique proteins from a single glomerulus and over 5k proteins from 6-10 human glomeruli. By snRNA-Seq, libraries were generated per single biopsy core, with glomerular and tubular populations represented. Despite normal histology, iNS patient-derived nuclei enriched in select proximal tubule and glomerular cell clusters, with altered gene signatures including increased cellular transport function and extracellular matrix accumulation, and reduced podocyte function.
We demonstrate that glomerular proteomic markers can be captured from 6 glomeruli from a human FFPE biopsy section. Key renal cell types are detectable by snRNA-Seq from standard clinical biopsy core, sufficient to detect differential cell composition and gene expression in disease tissue. This platform will be applied to identify novel therapeutic targets in kidney disease.
- Commercial Support – Novo Nordisk