Single-cell analysis of angiotensin-converting enzyme II expression in human kidneys and bladders reveals a potential route of 2019 novel coronavirus infection
In 2019, a novel coronavirus (2019-nCoV), later named SARS-CoV-2, emerged in Wuhan, China, and rapidly spread worldwide, causing the coronavirus disease 2019 (COVID-19) pandemic. While the primary symptoms of COVID-19 are respiratory, emerging evidence suggests that the virus can affect multiple organ systems, including the kidneys. Acute kidney injury (AKI) has been observed in a subset of COVID-19 patients, raising questions about the potential routes of viral infection in the urinary system. This study investigates the expression of angiotensin-converting enzyme II (ACE2), the receptor for SARS-CoV-2, in human kidneys and bladders using single-cell RNA sequencing (scRNA-Seq) to explore the mechanisms of kidney damage and potential viral entry routes in the urinary system.
Background and Significance
The 2019-nCoV, which shares 88% genomic similarity with bat-derived SARS-like coronaviruses, binds to ACE2 for cellular entry, similar to SARS-CoV. ACE2 is widely expressed in various tissues, including the lungs, kidneys, and bladder. While respiratory symptoms dominate COVID-19, AKI has been reported in 3-10% of hospitalized patients, with some cases progressing to renal failure. The detection of SARS-CoV-2 in urine samples further suggests the virus’s potential to infect the urinary system. However, the mechanisms underlying kidney damage and the role of ACE2 in urinary tract infection remain unclear.
This study leverages scRNA-Seq to profile ACE2 expression in healthy human kidneys and bladders, identifying cell types that may serve as viral targets. By analyzing co-expressed genes and functional pathways, the study provides insights into the potential routes of SARS-CoV-2 infection in the urinary system and the molecular mechanisms of kidney injury.
Methods
Data Acquisition and Processing
Public scRNA-Seq datasets from healthy kidneys and bladders were obtained from the Gene Expression Omnibus (GEO). Kidney data from three donors (GSE131685) and bladder data from three bladder cancer patients (GSE108097) were analyzed. For kidney samples, downstream analysis was performed using the Seurat package, with normalization, scaling, and clustering based on the top 2,000 variable genes. The bladder dataset was integrated using Harmony to remove batch effects, followed by similar normalization and clustering steps.
Kidney Sample Processing
Kidney biopsy samples were obtained from living donors and processed immediately. Tissue dissociation was performed using GEXSCOPE® Tissue Preservation Solution and dissociation buffer, followed by red blood cell lysis and cell counting. Single-cell libraries were prepared and sequenced using established protocols.
Co-expression and Functional Analysis
Co-expressed genes with ACE2 were identified using Pearson correlation coefficients, with genes showing r > 0.1 and P < 0.01 considered significant. Pathway enrichment analysis was performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Protein-protein interaction (PPI) networks were constructed using STRINGdb and visualized with Cytoscape.
Results
ACE2 Expression in Kidneys
Analysis of public kidney datasets revealed that ACE2 was predominantly expressed in proximal tubule (PT) cells, including proximal convoluted tubule (PCT) and proximal straight tubule (PST) cells. In three published datasets, ACE2 expression was detected in 5.12%, 5.80%, and 14.38% of PCT, PT, and PST cells, respectively. Validation using two independent healthy kidney samples confirmed ACE2 expression in 12.05%, 6.79%, and 10.20% of PCT, PT, and PST cells, respectively. Other nephron cell types, such as collecting duct and distal tubule cells, showed minimal ACE2 expression.
ACE2 Expression in Bladders
In the bladder dataset, ACE2 expression was low but detectable in epithelial cells, particularly umbrella cells (1.28%) and intermediate cells (0.25%). Other bladder cell types, including basal and immune cells, were largely negative for ACE2.
Functional Analysis of Co-expressed Genes
Co-expression analysis identified genes significantly correlated with ACE2 in PT and umbrella cells. These genes were enriched in membrane-related cellular components, including brush border membranes, and pathways such as the renin-angiotensin system, PT bicarbonate reclamation, and mineral absorption. PPI networks revealed interactions between ACE2 and co-expressed genes, suggesting potential roles in viral entry and kidney function.
Discussion
AKI in COVID-19 and SARS
AKI has been reported in COVID-19 patients, with a prevalence of 3-10% in hospitalized cases. Similar observations were made during the SARS outbreak, where 6.7% of patients developed AKI, with a high mortality rate. The detection of SARS-CoV-2 in urine and kidney tissues suggests direct viral infection of renal epithelial cells, potentially mediated by ACE2. The high expression of ACE2 in PT cells aligns with the observed kidney injury in COVID-19 patients.
ACE2 Expression in the Urinary System
The study demonstrates that ACE2 is expressed in specific cell types in the kidneys and bladders, with higher expression in PT cells compared to bladder epithelial cells. This finding suggests that the kidneys may be more susceptible to SARS-CoV-2 infection than the bladder. The detection of ACE2 in umbrella cells, which line the bladder lumen, supports the possibility of viral shedding in urine.
Implications for Clinical Practice
The findings highlight the need for monitoring kidney function in COVID-19 patients, particularly those with severe disease. The presence of ACE2 in renal and bladder epithelial cells underscores the potential for urinary transmission of SARS-CoV-2, warranting precautions in handling urine samples from infected patients.
Limitations
The study is limited by the use of healthy tissue samples, which may not fully reflect ACE2 expression patterns in COVID-19 patients. Additionally, the lack of experimental validation and patient-derived data restricts the ability to draw definitive conclusions about viral infection mechanisms in the urinary system.
Conclusion
This study provides bioinformatics evidence of ACE2 expression in human kidneys and bladders, identifying PT and umbrella cells as potential targets for SARS-CoV-2 infection. The findings suggest that kidney injury in COVID-19 may result from direct viral infection of renal epithelial cells, mediated by ACE2. Further research is needed to validate these findings and explore the mechanisms of urinary tract infection and kidney damage in COVID-19 patients.
doi.org/10.1097/CM9.0000000000001439
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