Gene Expression
Nebulas
Gene Expression NebulasSummary: Background: Acute kidney injury (AKI) is a common sequela of infection with SARS-CoV-2 and contributes to the severity and mortality from COVID-19. Here, we tested the hypothesis that kidney alterations induced by COVID-19-associated AKI could be detected in cells collected from urine. Methods: We performed single-cell RNA sequencing (scRNAseq) on cells recovered from the urine of eight hospitalized COVID-19 patients with (n=5) or without AKI (n=3) as well as four non-COVID-19 AKI patients (n=4) to assess differences in cellular composition and gene expression during AKI. Results: Analysis of 30,076 cells revealed a diverse array of cell types, most of which were kidney, urothelial, and immune cells. Pathway analysis of tubular cells from patients with AKI showed enrichment of transcripts associated with damage-related pathways compared to those without AKI. ACE2 and TMPRSS2 expression were highest in urothelial cells amongst cell types recovered. Notably, in one patient we detected SARS-CoV-2 viral RNA in urothelial cells. These same cells were enriched for transcripts associated with anti-viral and anti-inflammatory pathways. Conclusions: We successfully performed scRNAseq on urinary sediment from hospitalized patients with COVID-19 to noninvasively study cellular alterations associated with AKI and established a dataset that includes both injured and uninjured kidney cells. Additionally, we provide preliminary evidence of direct infection of urinary bladder cells by SARS-CoV-2. The urinary sediment contains a wealth of information and is a useful resource for studying the pathophysiology and cellular alterations that occur in kidney diseases.
Overall Design: Single-cell RNA sequencing of cells obtained from urine of patients with COVID-19. There are 12 total samples from 12 separate patients, 5 with COVID19 AKI, 3 with COVID19 without AKI, and 4 with non COVID19 AKI
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| Extract Protocol: | All steps were performed on ice. Urine was collected as either a voided specimen or from a urinary catheter. Samples were immediately transferred to a biosafety level (BSL) 2+ laboratory for processing. Urine samples were transferred to a 50mL conical tube and centrifuged at 1000x g for 10 minutes at 4 degrees celsius. Cell pellets were washed with ice-cold PBS, filtered through a 40µm filter, and centrifuged again. Live cells were purified using the MACS Debris Removal Kit (Miltenyi Biotec) followed by the EasySep Annexin V Dead Cell Removal Kit (StemCell). Briefly, cells were resuspended in ice-cold PBS and mixed with debris removal solution. Cold PBS was overlaid on the mixture and centrifuged at 300x g for 10 minutes at 4 degrees celsius. The top two phases were aspirated and then remaining cells were washed with PBS. Cells were resuspended and mixed with Dead Cell Removal Cocktail, Biotin Selection Cocktail, then RapidSpheres before separation in an EasySep magnet. Cells were washed and resuspended in 52µL of PBS (no calcium or magnesium) + .04% BSA (Fisher Scientific) for scRNAseq processing. Purified cells were immediately processed using the Chromium 3’ Single Cell RNA sequencing kit (10X Genomics) according to the manufacturer’s instructions. |
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| Molecule Type: | polyA(+) RNA |
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| Library Layout: | PAIRED |
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| Platform: | ILLUMINA |
| Instrument Model: | Illumina NovaSeq 6000; Illumina NextSeq 500 |
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| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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