SARS-CoV-2 infects the human kidney and drives fibrosis in kidney organoids

Abstract

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human-induced pluripotent stem-cell-derived kidney organoids with SARS-CoV-2. Single-cell RNA sequencing indicated injury and dedifferentiation of infected cells with activation of profibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in long COVID.

Keywords: COVID-19; SARS-CoV-2; chronic kidney disease; fibrosis; human iPSC kidney organoids; kidney injury; protease blocker.

Graphical abstract

Figure 1

SARS-CoV-2 is present in COVID-19 patient kidney cells and induces fibrosis (A) SARS-CoV-2 nucleocapsid protein (NP, red, perinuclear), ACE2 (green), and LTL (blue) expression in human COVID-19 kidney biopsy tissue. Scale bars, 10 μm. (B–G) SARS-CoV-2 NP (B–D, arrowhead, red), ACE2 (B, C, and F, green), and LTL (B, C, and E, blue) expression in human COVID-19 autopsy kidney tissue, (G) autopsy control. Scale bars (C–F), 10 μm. (H and I) KIM-1 (green), LTL (blue), and NKCC2 (red) expression in human COVID-19 patient kidney biopsy tissue, (I) nephrectomy control. (K and J) Interstitial fibrosis (collagen fibers in blue) is enhanced in (K) COVID-19 autopsy tissues compared with (J) nephrectomy controls, as quantified based on Masson’s trichrome staining. (L) COVID-19 patient (n = 62) and control cohort (n = 57) characteristics. For details refer to Table S1. (M) ImageJ quantification of collagen expression in Masson’s trichrome-stained COVID-19 patient kidney autopsy tissue compared with control nephrectomy tissue and control ARDS/influenza patient autopsy kidney tissue. (N) Collagen expression quantification in COVID-19 patient kidney autopsy tissue including patients without CKD, diabetes, and hypertension only. (O) Comparing COVID-19 patients kidney autopsy tissue with or without pre-existing CKD to respective control cohorts with or without CKD. In Figure S1, representative staining results of other patients included in this study as well as non-COVID-19 autopsy controls are shown. In (M)-(O), data are presented as mean and SD. Scale bars, 50 μm, unless stated otherwise.^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001. See also Figure S1 and Table S1.

Figure 2

SARS-CoV-2 is present in COVID-19-patient kidney cells on single-cell level and induces ECM remodeling (A) UMAP overview of clusters detected in snRNA-seq data of one human COVID-19 patient kidney tissue sample. (B) Integrated UMAP of COVID-19-patient kidney tissue snRNA-seq and control snRNA-seq from a public dataset (Muto et al., 2021). (C) Coverage plot of the SARS-CoV-2 genome in COVID-19 patient kidney snRNA-seq data. (D) Targeted sequencing (tapSeq)-based mapping of SARS-CoV-2-containing cells (red dots) per cluster. (E) Extracellular matrix (ECM) gene set enrichment analysis (GSEA) shows ECM upregulation in COVID-19 versus control kidney snRNA-seq data. (F) PROGENy pathway analysis reveals fibrosis-related pathway upregulation in COVID-19 versus control snRNA-seq data. (G) Cell-cell interaction bar plot depicting cell clusters with the highest and lowest interaction scores in COVID-19 versus control kidney snRNA-seq datasets. (H) Ligand-receptor analysis shows fibrosis-related pathways upregulated in proximal tubular epithelial cells (PTEC), fibroblasts, and podocytes. (I) Sankey plot illustrating upregulated ligand-receptor interactions from proximal tubular cells to fibroblasts .^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. See also Figure S2 and Table S2.

Figure 3

SARS-CoV-2 infects human iPSC-derived kidney organoid cells and stimulates ECM deposition (A and B) Schematic illustration of the iPSC-derived kidney organoids culture. (C) Segmented patterning in human kidney organoids shows the presence of podocytes (NPHS1, purple), proximal tubules (LTL, blue), distal-like (ECAD, green), and collecting duct-like (ECAD plus GATA3 [red] costaining) structures. Scale bars, 200 μm. (D) ACE2 protein expression (green) is present in organoid proximal tubules (LTL, blue). Scale bars, 20 μm. (E) COVID-19 viral RNA detection in infected kidney organoids. For the mock infection, the same type of medium was used as that in which virus was propagated and added as the same percentage as in the virus-infected organoids. Data are given as mean and SD from 3 independent experiments each performed with 2 biological replicates. ^∗∗∗p ≤ 0.001 (F) mRNA transcript expression of SARS-CoV-2 open reading frame (ORF, brown stain, arrow heads) in podocytes using BROWN Assay In Situ Hybridization (BRISH). Scale bars, 50 μm. (G) Periodic acid-Schiff stain of the BRISH positive podocyte cluster shown in (F). Scale bars, 50 μm. (H and I) (H) mRNA transcript expression of ACE2, SARS-CoV-2 antisense, and (I) TMPRSS2 in proximal tubules using FISH. Scale bars, 20 μm. (J and K) SARS-CoV-2 nucleocapsid protein detection (green) in proximal tubules (LTL, blue) in (J) mock-infected and (K) infected kidney organoids. Scale bars, 50 μm. (L–N) Immuno-based correlative light and electron microscopy (CLEM) analysis of a (L) SARS-CoV-2 nucleocapsid protein-positive mesenchymal cell. Scale bars, 10 μm. (M and N) TEM ultrastructure analysis of viral particles (60–70 nm) in the corresponding SARS-CoV-2 nucleocapsid protein-positive mesenchymal cell as shown in (L). (M) Scale bars, 500 nm; (N) scale bars, 100 nm. In Figure S3, the sequential CLEM workflow of a cell of interest is shown. (O and P) 3D FIB-SEM segmentation showing viral particles (orange) in a vacuole (magenta) in close proximity to the nucleus (blue). In (O), a total volume of 5 × 7 × 1.7 μm³ is shown. Voxel size: 5 nm. (P) 3D reconstruction of the stack presented in (O). The vacuole (purple) dimensions are ∼2 × 1 × 1.7 μm³. (Q–U) Collagen I expression quantification of (R) mock, (S) SB431542 (10 μM), (T) SARS-CoV-2 (^∗ increased collagen I [green]), and (U) SARS-CoV-2 plus SB431542-treated organoids. Scale bars, 20 μm. Data are mean and SD from five independent experiments, each performed with two biological replicates, of which two experiments were supplemented with the TGFb inhibitor SB431542 (10 μM). ^∗p < 0.05, ^∗∗p < 0.01. (V–X) Masson’s trichrome staining of (V) mock and (W) SARS-CoV-2-infected organoids (^∗ collagenous extracellular matrix, [blue]), (X) quantification. Scale bars, 20 μm. Data are given as mean and SD from four independent experiments each performed with two biological replicates. ^∗p < 0.05. See also Figure S3.

Figure 4

Single-cell-level resolution reveals 15 distinct cell clusters and detection of the SARS-CoV-2 genome in SARS-CoV-2-infected human iPSC-derived kidney organoids (A) UMAP integration of 27,754 single cells of human iPSC-derived kidney organoids results in 15 different cell clusters: mesenchyme 1, mesenchyme 2, PT (proximal tubule) cells, podocytes, LP (loop progenitor) cells, 6 neural cell clusters (neural cells 1–5 and NP [neural progenitor] cells), and 4 muscle cell clusters (muscle cells 1–4). (B) UMAP visualization of cell cycle phases. Cells in phase G1 are illustrated in red, those in G2M stage in green, and S-phase cells in blue. (C) Heatmap illustrating a selection of the top 20 differentially expressed (DE) genes per cluster. The full list of the top 20 DE genes per cluster corresponding to this heatmap is available in Table S3. (D) UMAP depicting proximal tubule cells (PT cells) and podocytes by marker genes (both populations are highlighted by black dashed lines; PT cells marker genes: EPCAM, KRT18, LRP2 [top 119], ABCC4 [top 201], and SLC22A5 [top 416]; podocyte marker genes: NPHS2 and PODXL). Gray dots represent all non-PT and nonpodocyte cells. (E) Cell proportion analysis of the 2 mock-infected and 2 SARS-CoV-2-infected samples. (F) scRNA sequencing of control and SARS-CoV-2-infected iPSC-derived kidney organoids resulted in the illustrated cell-type distribution per sample. In all four samples, we could identify 15 distinct cell populations (mesenchyme 1, mesenchyme 2, PT (proximal tubule) cells, podocytes, LP (loop progenitor) cells, 6 neural cell clusters (neural cells 1–5 and NP [neural progenitor] cells), and 4 muscle cell clusters (muscle cells 1–4). (G) Coverage plot of reads aligned to the SARS-CoV-2 genome in infected sample 2 (The respective plot for sample 1 is found in Figure S4K). (H) SARS-CoV-2 reads per single cell per cluster. The color gradient ranges from gray (0 reads per cell) to dark red (119 reads per cell). An overview of viral reads and number of infected cells per cluster and sample is shown in Table S4. See also Figure S4 and Tables S3 and S4.

Figure 5

Pathway and ligand-receptor analysis in control versus SARS-CoV-2-infected human iPSC-derived kidney organoids reveal upregulation of fibrosis-related pathways and corresponding cellular crosstalk (A) Top 10 most prominent genes expressed per mesenchymal (mesenchyme 1 and 2) and kidney cell clusters (PT [proximal tubule] cells, podocytes, and LP [loop progenitor] cells), compared between cells where we could detect viral transcription and cells of the same cluster without detectable viral transcription. Here, we only consider cells from the SARS-CoV-2-infected organoids. (B) Heatmap illustrating upregulated pathways derived from the KEGG database in control versus SARS-CoV-2-infected human iPSC-derived kidney organoids. (C) PROGENy violin plots showing selected pathway gene sets in control (gray) versus SARS-CoV-2 (purple)-infected human iPSC-derived kidney organoids. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. (D) DoRothEA heatmap showing upregulated (red) and downregulated (blue) transcription factor activity in control versus SARS-CoV-2-infected human iPSC-derived kidney organoids. (E) Map of differential cell-cell interaction (CCI) network uncovering cellular crosstalk between select clusters in scRNA sequencing data comparing SARS-CoV-2-infected iPSC-derived kidney organoids to untreated control iPSC-derived kidney organoids. (F–H) Bar graphs highlighting the top-ranked (F) ligands in proximal tubule cells, (G) podocytes, and (H) receptors in mesenchyme 1 cells. (I and J) Sankey diagrams showing ligand-receptor pairs between (I) proximal tubule cells and mesenchyme 1 cells and (J) podocytes and mesenchyme 1 cells. See also Figure S5.

Figure 6

SARS-CoV-2 uptake is inhibited in human iPSC-derived kidney organoids by a protease inhibitor (A) Chemical structure of MAT-POS-b3e365b9-1. (B) Dose-response curve in Vero E6 cells infected with SARS-CoV-2 (isolate BavPat1) and treated with the indicated concentrations of MAT-POS-b3e365b9-1 or DMSO at 24 h after infection (hpi). Data are given as mean and SD from two independent experiments. (C–E) Intracellular viral RNA at 48 and 96 hpi, and (D and E) plaque assay on Vero E6 cells of the apical medium at 96 hpi of kidney organoids infected with SARS-CoV-2 in the presence of 10-μM MAT-POS-b3e365b9-1 or DMSO. Data are given as mean and SD from a representative experiment of two independent experiments, each performed with two biological replicates. Intracellular viral RNA levels in (C) were normalized to expression of the β-actin housekeeping gene. ∗∗p < 0.01, ∗∗∗p < 0.001.