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. 2021 Nov 5;3(1):28-36.
doi: 10.34067/KID.0005522021. eCollection 2022 Jan 27.

Single-Cell RNA Sequencing of Urinary Cells Reveals Distinct Cellular Diversity in COVID-19-Associated AKI

Matthew D Cheung  1   2   3 , Elise N Erman  1   2   3 , Shanrun Liu  4 , Nathaniel B Erdmann  5 , Gelare Ghajar-Rahimi  1   2   3 , Kyle H Moore  1   2   3 , Jeffrey C Edberg  4 , James F George  2   3 , Anupam Agarwal  1   2
Affiliations

Single-Cell RNA Sequencing of Urinary Cells Reveals Distinct Cellular Diversity in COVID-19-Associated AKI

Matthew D Cheung et al. Kidney360. .

Abstract

Background: 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 patients with COVID-19 with (n=5) or without AKI (n=3) as well as four patients with non-COVID-19 AKI (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 with those without AKI. ACE2 and TMPRSS2 expression was highest in urothelial cells among 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 antiviral 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.

Keywords: COVID-19; SARS-CoV-2; acute kidney injury; acute kidney injury and ICU nephrology; basic science; single-cell RNA sequencing; urine.

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Conflict of interest statement

A. Agarwal reports consultancy agreements with Akebia Therapeutics (served on an expert panel to review new therapeutics on the basis of the hypoxia-inducible factor pathway for AKI), Dynamed (reviewed content related to AKI for Dynamed and reviewed and updated materials prepared by the Dynamed editorial team for AKI topics), and Reata Pharmaceuticals (served as a consultant); ownership interest in Goldilocks Therapeutics, Inc.; research funding from the Genzyme/Sanofi Fabry fellowship award; and honoraria from Akebia Therapeutics, Emory, the University of Southern California, and Vanderbilt. A. Agarwal also reports scientific advisor or membership as an editorial board member for American Journal of Physiology–Renal Physiology, Kidney International, and Laboratory Investigation; as an advisory board member of Goldilocks Therapeutics, Inc. (a New York–based company investigating the delivery of drugs in the kidney using nanotechnology for acute kidney disease and CKD); as an external evaluation panel member for the Kidney Precision Medicine Program; and as an advisory board member of Alpha Young, LLC and Angion. All remaining authors have nothing to disclose.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
Single-cell RNA sequencing of urine from patients with coronavirus disease 2019 (COVID-19) with and without AKI and patients with non–COVID-19 AKI. (A) Integrated uniform manifold approximation and projection (UMAP) plot of 30,076 cells sequenced from patient urine sediment. (B–D) UMAP plot subsetted by COVID-19 and AKI status showing the difference in collected cell populations. Cell-type labels are the same. (E) Heat map of differentially expressed genes of integrated clusters. (F) Dot plot of known marker genes for identification of cells. B, B lymphocyte; CD, collecting duct; IC, intercalated cell; KRM, kidney resident macrophage; LOH, loop of Henle; Mac, macrophage; Neut, neutrophil; Podo, podocyte; PT, proximal tubule; RBC, red blood cell; SMC, smooth muscle cell; T, T lymphocyte; Uro, urothelial.
Figure 2.
Figure 2.
Enrichment of damage-related pathways in cells from patients with AKI. Gene Set Enrichment Analysis of each cluster of cells from the integrated UMAP for pathways, including (A) hallmark reactive oxygen species pathways, (B) hallmark apoptosis pathways, and (C) hallmark DNA repair pathways in patients with COVID-19 AKI (left panels), COVID-19 no AKI controls (center panels), and patients with non–COVID-19 AKI (right panels). Cluster identifications are as follows: 1, urothelial; 2, urothelial; 3, urothelial; 4, macrophage; 5, MHC class 2–positive neutrophil; 6, loop of Henle; 7, neutrophil; 8, collecting duct; 9, urothelial; 10, umbrella; 11, neutrophil; 12, collecting duct; 13, kidney resident macrophage; 14, neutrophil; 15, urothelial; 16, cycling; 17, proximal tubule; 18, proximal tubule; 19, smooth muscle cell; 20, intercalated cell; 21, urothelial; 22, T cell; 23, podocyte; 24, red blood cell; 25, macrophage; 26, B cell; 27, macrophage; 28, red blood cell.
Figure 3.
Figure 3.
Detection of direct infection of urothelial cells by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). (A) Violin plots of ACE2, TMPRSS2, and SARS-CoV-2 gene expression among sequenced cell types. Cell types with identified SARS-CoV-2 gene expression are identified in blue to identify overlap between SARS-CoV-2 receptor and gene expression. (B) Individual analysis of patient 7: UMAP plots and ACE2 and SARS-CoV-2 expression identifying the presence of SARS-CoV-2 gene expression within urothelial cells. (C) Urinalysis laboratory values from urine samples taken from patient 7 2 days prior and 12 days after urine collection for single-cell RNA sequencing. (D) Gene enrichment analysis using the WebGestaltR Biologic Process to identify upregulated pathways in the urothelial cells of patient 7. Pathways involved in viral infection and immune response are shown in blue with a false discovery rate (FDR) of ≤0.05. Sq Epi, squamous epithelial cells; Leuk Est, leukocyte esterase; lmm, immune cells; Neg, negative; SG, specific gravity; WBC, white blood cell.
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