A specific molecular signature in SARS-CoV-2-infected kidney biopsies

Abstract

Acute kidney injury is one of the most important complications in patients with COVID-19 and is considered a negative prognostic factor with respect to patient survival. The occurrence of direct infection of the kidney by SARS-CoV-2, and its contribution to the renal deterioration process, remain controversial issues. By studying 32 renal biopsies from patients with COVID-19, we verified that the major pathological feature of COVID-19 is acute tubular injury (ATI). Using single-molecule fluorescence in situ hybridization, we showed that SARS-CoV-2 infected living renal cells and that infection, which paralleled renal angiotensin-converting enzyme 2 expression levels, was associated with increased death. Mechanistically, a transcriptomic analysis uncovered specific molecular signatures in SARS-CoV-2-infected kidneys as compared with healthy kidneys and non-COVID-19 ATI kidneys. On the other hand, we demonstrated that SARS-CoV-2 and hantavirus, 2 RNA viruses, activated different genetic networks despite triggering the same pathological lesions. Finally, we identified X-linked inhibitor of apoptosis-associated factor 1 as a critical target of SARS-CoV-2 infection. In conclusion, this study demonstrated that SARS-CoV-2 can directly infect living renal cells and identified specific druggable molecular targets that can potentially aid in the design of novel therapeutic strategies to preserve renal function in patients with COVID-19.

Keywords: Apoptosis; COVID-19; Molecular pathology; Nephrology.

Figure 1. SARS-CoV-2 infects kidney cells.

(A) Results of SARS-CoV-2 FISH on uninfected and SARS-CoV-2–infected Vero cells (left) and on human lung tissue of healthy and COVID-19 patients (right). (B) Results of SARS-CoV-2 FISH on control kidneys (healthy and ATI of non–COVID-19 patients) and on a kidney from a patient with COVID-19. (C) Representative images of the different patterns of SARS-CoV-2 FISH detection in kidneys from patients with COVID-19. Of note, the first 2 images on the left show SARS-CoV-2 staining in tubules. The third image from the left shows SARS-CoV-2 staining in a glomerulus. The dotted lines outline the glomerular capsule. The fourth image (right) shows SARS-CoV-2 staining in interstitial cells. The dotted lines outline the tubular basal membrane. White arrows show positive cells. Positive-strand RNA was labeled with Cy5 (red), and nuclei in blue (DAPI). Scale bars in all panels: 100 μm.

Figure 2. SARS-CoV-2 renal infection elicits a specific molecular signature.

(A) PCA of the top 500 most variable genes in SARS-CoV-2 FISH-positive and FISH-negative kidneys and healthy kidneys. (B) Heatmap representing K-means analysis of DEGs comparing healthy kidneys and SARS-CoV-2 FISH-positive and SARS-CoV-2 FISH-negative COVID-19 kidneys. (C) Dot plot of the cluster profiling analysis from K-means comparing healthy kidneys and SARS-CoV-2 FISH-positive and SARS-CoV-2 FISH-negative COVID-19 kidneys. (D) Bar plot of the top 15 normalized enriched score–ranked gene sets (hallmark, GSEA) with q < 0.001 in COVID-19 kidneys compared with healthy kidneys.

Figure 3. SARS-CoV-2 renal infection elicits a specific molecular signature compared with non–COVID-19 ATI kidneys.

(A) Heatmap representing K-means analysis with the corresponding dot plot of the cluster profiling of DEGs comparing healthy kidneys, SARS-CoV-2 FISH-positive, SARS-CoV-2 FISH-negative, and non–COVID-19 ATI kidneys. (B) Bar plot of the top 5 combined score-ranked gene sets (hallmark, GSEA) from cluster 2 of the K-means clustering analysis comparing healthy kidneys, SARS-CoV-2 FISH-positive, SARS-CoV-2 FISH-negative, and non–COVID-19 ATI kidneys. (C) Bar plot of the top 10 normalized enriched score–ranked gene set (hallmark, GSEA) of FISH-positive COVID-19 kidneys compared with non–COVID-19 ATI kidneys (left panel). Hallmark IFN-α enrichment plot from GSEA comparing FISH-positive COVID-19 kidneys with non–COVID-19 ATI kidneys (right panel).

Figure 4. SARS-CoV-2 renal infection elicits a specific molecular signature compared with hantavirus kidneys.

(A) Heatmap representing K-means analysis with the corresponding dot plot of the cluster profiling of DEGs comparing healthy kidneys and SARS-CoV-2 FISH-positive and hantavirus kidneys. (B) Bar plot of the top 5 combined score-ranked gene sets (hallmark, GSEA) from clusters 2 and 3 of the K-means clustering analysis comparing healthy kidneys and SARS-CoV-2 FISH-positive and hantavirus kidneys.

Figure 5. XAF1 is a critical target of renal SARS-CoV-2.

(A) Volcano plot of DEGs comparing FISH-positive COVID-19 kidneys with non–COVID-19 ATI kidneys. (B) Comparative XAF1 gene expression in healthy kidneys, non–COVID-19 ATI kidneys, and FISH-positive and FISH-negative COVID-19 kidneys (left panel) and healthy kidneys, non–COVID-19 ATI kidneys, and FISH-positive and hantavirus kidneys (right panel). Tukey-Kramer test was applied to test the significance of the difference. ***P < 0.001. (C) Representative images of cleaved caspase-3 immunostaining in healthy kidneys and FISH-positive COVID-19 kidneys. (D) Hallmark apoptosis enrichment plot from GSEA comparing FISH-positive COVID-19 kidneys with healthy controls.