The orchestrated cellular and molecular responses of the kidney to endotoxin define a precise sepsis timeline

  1. Danielle Janosevic
  2. Jered Myslinski
  3. Thomas W McCarthy
  4. Amy Zollman
  5. Farooq Syed
  6. Xiaoling Xuei
  7. Hongyu Gao
  8. Yun-Long Liu
  9. Kimberly S Collins
  10. Ying-Hua Cheng
  11. Seth Winfree
  12. Tarek M El-Achkar
  13. Bernhard Maier
  14. Ricardo Melo Ferreira
  15. Michael T Eadon
  16. Takashi Hato  Is a corresponding author
  17. Pierre C Dagher  Is a corresponding author
  1. Department of Medicine, Indiana University School of Medicine, United States
  2. Department of Pediatrics and the Herman B. Wells Center, Indiana University School of Medicine, United States
  3. Department of Medical and Molecular Genetics, Indiana University School of Medicine, United States
  4. Roudebush Indianapolis Veterans Affairs Medical Center, United States
9 figures, 1 table and 7 additional files

Figures

Figure 1 with 2 supplements
ScRNA-seq identifies various renal cell populations.

(A) Integrated UMAP of kidney cell clusters from control and LPS-treated mice (0, 1, 4, 16, 27, 36, and 48 hr after LPS injection). Actual anatomical layout of kidney nephronal segments is shown …

Figure 1—figure supplement 1
Cluster-defining markers across the endotoxemia timeline.

(A) Integrated UMAP of kidney cell clusters showing both assigned identity and original cluster number from control and LPS-treated mice (0, 1, 4, 16, 27, 36, and 48 hr after LPS injection). (B–C) …

Figure 1—figure supplement 2
Characterization of proliferating cells.

(A) Feature plots of Jun and Fos, markers for dissociation-induced stress response. Note that proliferating cells (red circles) showed minimal expression of Jun and Fos. (B) Mice were injected with …

Figure 2 with 2 supplements
Integration of scRNA-seq and spatial transcriptomics localizes subtypes of S3 proximal tubules.

(A) Violin plots of S3T2 defining markers. (B) Integration of spatial transcriptomics and scRNA-seq. Spatial transcriptomics were performed on a slice of mouse kidney. This yielded seven clusters …

Figure 2—figure supplement 1
Spatial transcriptomics validation.

(A–B) Spatial transcriptomics of murine kidney showing unsupervised clustering (b-left panel) and expanded clustering after integration with single cell data (b-right panel). Insets of A show gene …

Figure 2—figure supplement 2
Cellular expression of the RAS axis along the endotoxemia timeline.

Feature plots showing expression of components of the RAS axis at specified time points. Labeled in the upper left panel are the segments of the proximal tubule, and stromal cells which express …

Figure 3 with 1 supplement
Endotoxemia induces dynamic changes in renal immune cell composition, pseudotime states and RNA velocity.

(A) Integrated UMAP of the immune cell clusters from control and LPS-treated mice (0, 1, 4, 16, 27, 36, and 48 hr after LPS injection). Other one and Other two are Cd45+ cells with mixed epithelial …

Figure 3—figure supplement 1
Immune cell subset characteristics.

(A) RNA velocity analysis reveals two distinct subfields within the Mϕ-A cluster. The subfield circled in red showed expression of M2 macrophage-related genes at later time points (B). (C) Violin …

Figure 4 with 2 supplements
Pseudotime and velocity field analyses identify cell-specific phenotypic changes along the endotoxemia timeline.

(A) Cell trajectory analysis for S1, S3, and endothelial cells shown at indicated time points. Highlighted in red circles are significant state transitions in respective cell types. The last cell …

Figure 4—figure supplement 1
Transporter gene expression across the nephron during endotoxemia.

Dot plots of select transporter genes shown at 0 hr (A) and 16 hr (B).

Figure 4—figure supplement 2
Similarity analysis between bulk and single-cell RNA-seq data.

(A) Jaccard similarity index analysis performed on the current single-cell RNA-seq data and publicly available bulk kidney transcriptomic dataset (GSE30576). Differentially expressed genes over …

Figure 5 with 3 supplements
Endotoxemia induces an organ-wide host defense phenotype in the kidney.

(A) Heatmaps of select cell types with top 100 differentially expressed genes across the endottoxemia timeline (0–48 hr). Select genes are shown for each cell type. (B) Time dependent enrichment of …

Figure 5—figure supplement 1
Comparisons of transcriptomic profiles of selected cell types across the endotoxemia timeline.

(A) Venn diagram showing differentially expressed genes across time (0–48 hr) for indicated cell types. (B) Heatmaps of genes involved in coagulation, complement and arachidonic acid related …

Figure 5—figure supplement 2
S3T2 GO terms.

Time dependent enrichment of gene ontology terms for S3T2 cells. GO terms are sorted in order of statistical significance. GO, gene ontology biological processes.

Figure 5—figure supplement 3
Representative images of immunohistochemical staining for NF-κB are shown under indicated conditions.

Insets point to magnified views of select areas. At baseline, the expression of NF-κB is minimal in all cell types and primarily cytoplasmic. One hour after LPS, diffuse nuclear staining for NF-κB …

Figure 6 with 1 supplement
Endotoxemia alters cellular crosstalk causing time-specific global communication failure.

(A) Schematic description of a cell–cell communication circular plot. Dots in the outer track of the circle represent specific ligands or receptors and are positioned identically for all cell types. …

Figure 6—figure supplement 1
Expanded cell–cell communication examples.

(A) Feature plots illustrating cell and time-dependent expression changes of select receptor ligand pairs (shown at 0 hr unless otherwise specified). Schematic illustrates specific receptor–ligand …

Figure 7 with 2 supplements
Global communication failure is accompanied by increased activity of genes involved in recovery.

(A) SCENIC-derived heatmap of regulons for S1 tubules. Highlighted are select transcription factors with active regulons at the 16 hr time point. (B) Gene ontology pathway enrichment analysis …

Figure 7—figure supplement 1
Characteristics of subjects.

(A) Age distribution of subjects (22 subjects are sorted based on gene expression as shown in main Figure 7D and E. p=0.223). (B) Distribution of serum creatinine changes from baseline to peak …

Figure 7—figure supplement 2
Serum biomarkers and chemokine/cytokine levels.

(A) Serum creatinine levels at indicated time points after 5 mg/kg LPS i.v. n = 3, *p<0.05 vs other time points. (B, C) Serum cystatin C and NGAL levels. n = 4. (D–F) Comparison of serum …

Author response image 1
Bulk kidney microarray data.
Author response image 2
Timeline of murine LPS model.

(A) Adapted from Figure 4. Single-cell RNA-seq data demonstrating overall time-dependent gene expression changes in proximal tubules after LPS challenge. Proximal tubules exhibit major phenotypic …

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Commercial assay, kitRNAscope probe-Mm-AgtAdvance Cell DiagnosisCat. No. 426941
Commercial assay, kitRNAscope probe-Mm-Aqp1Advance Cell DiagnosisCat. No. 504741-C2
Commercial assay, kitMilliplex MAP Mouse Cytokine/Chemokine Magnetic Bead Panel–Premixed 32 PlexMilliporeCat. No. MCYTMAG-70K-PX32
Commercial assay, kitAnnexin V dead cell removal kitStem Cell TechnologiesCat. No. 17899
Commercial assay, kitMulti-Tissue Dissociation Kit 2Miltenyi BiotecCat. No. 130-110-203
Commercial assay, kitChromium Single Cell 3' Library and gel bead kit10x GenomicsCat. no. 1000121
Commercial assay, kitNovaSeq 6000 S1 reagent kitIlluminaCat. No. 20012865
Commercial assay, kitVisium Spatial Gene Expression library preparation slide10x GenomicsCat. No. 1000200
Chemical compound, drugRed blood cell lysing buffer Hybri-MaxSigmaCat. No. R7757
AntibodyNFkB P65 (D14E12 rabbit monoclonal)Cell SignalingCat. 8242S
Chemical compound, drugCldUSigmaCat. C6891
Strain, strain background (Escherichia coli)LPS E. coli serotype o111:B4SigmaCat. No. L2630
Lot No. 095M4163V
Biological samplesHuman renal biopsy bulk AKI RNAseq dataPMID:30507610GEO: GSE122274
Software, algorithmMonocleCao et al., 2019PMID:30787437
Software, algorithmSeuratStuart et al., 2019; Butler et al., 2018RRID:SCR_016341https://satijalab.org/
Software, algorithmSCENICAibar et al., 2017PMID:28991892
Software, algorithmCellphone DBEfremova et al., 2020; Vento-Tormo et al., 2018https://www.cellphonedb.org/
Software, algorithmRNA velocityLa Manno et al., 2018PMID:30089906
Software, algorithmSingleRAran et al., 2019PMID:30643263
Software, algorithmHarmony and PalantirNowotschin et al., 2019; Setty et al., 2019PMID:30959515
PMID:30899105
Software, algorithmRR Project for Statistical ComputingRRID:SCR_001905http://www.r-project.org/
Commercial protocolRNAscope multiplex Fluorescent Reagent Kit v2Advance Cell Diagnosis Inc
OtherDead cell removal protocol using Annexin Vhttps://cdn.stemcell.com/media/files/pis/DX21956-PIS_1_0_1.pdf?_ga=2.34218465.1547447083.1547219505%E2%80%93776976877.1534951026Commercial protocol
OtherChromium Single Cell 3’ Reagent Kits V3 User Guidehttps://assets.ctfassets.net/an68im79xiti/51xGuiJhVKOeIIceW88gsQ/1db2c9b5c9283d183ff4599fb489a720/CG000183_ChromiumSingleCell3__v3_UG_Rev-A.pdfCommercial protocol
OtherDissociation of mouse kidney using the Multi Tissue Dissociation Kit 2https://www.miltenyibiotec.com/upload/assets/IM0015569.PDFCommercial protocol

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