Kidney intercalated cells are phagocytic and acidify internalized uropathogenic Escherichia coli

Kidney intercalated cells are involved in acid-base homeostasis via vacuolar ATPase expression. Here we report six human intercalated cell subtypes, including hybrid principal-intercalated cells identified from single cell transcriptomics. Phagosome maturation is a biological process that increases in biological pathway analysis rank following exposure to uropathogenic Escherichia coli in two of the intercalated cell subtypes. Real time confocal microscopy visualization of murine renal tubules perfused with green fluorescent protein expressing Escherichia coli or pHrodo Green E. coli BioParticles demonstrates that intercalated cells actively phagocytose bacteria then acidify phagolysosomes. Additionally, intercalated cells have increased vacuolar ATPase expression following in vivo experimental UTI. Taken together, intercalated cells exhibit a transcriptional response conducive to the kidney’s defense, engulf bacteria and acidify the internalized bacteria. Intercalated cells represent an epithelial cell with characteristics of professional phagocytes like macrophages.


Remove column from magnet and Elute c-KIT + Cells
ScRNAseq using 10x genomics platform a b Supplementary Figure 1: The enrichment of ICs from human kidney samples (a) E1 subunit of V-ATPase (magenta pseudo color from red) (asterisks, left panel) and cKIT (green, arrow, middle panel) are presented. C-KIT and the VATPase (E1 subunit) colocalized to the same cells (arrowheads, right panel) indicating that c-KIT represent a cell surface protein to target ICs for enrichment. Of note, c-KIT also stains stromal cells/telocytes and can intermittently stains some cells not positive for V-ATPase (E1 subunit) (green only cells, right panel) likely representing proximal tubules and loop of Henle cells seen in scRNAseq ( Figure  1). These aforementioned cell types may represent the c-KIT staining that did not localize with the V-ATPase E1 subunits. Additionally, some green autofluorescence by red blood cells during IF imaging can occur. To confirm the overall IC enrichment when c-KIT positive kidney cells are magnetic sorted, normal margins from mass resection samples from 4 distinct individuals (a-d) were tested and scatter plots with superimposed bar graphs are presented. Each dot represents a run in a duplicate well. ATP6V1B1 (IC marker), SLC4A1 (A-IC marker) and SLC26A4 (B-IC marker) mRNA expression was measured by RT-PCR. The c-KIT enriched IC fraction was compared to the c-KIT negative fraction. We had variable degrees of enrichment for ICs in general (ATPV1B1), A-ICs (SLC4A1) and B-ICs (SLC26A4). SLC26A4 was not detected in biopsy 2 (b) wells. Relative enrichment is presented from 4 distinct patient kidney tissue samples. Source data are provided as a Source Data File.
The 9 leading cell type markers identified on scRNAseq analysis for cluster O are presented. The data is presented with the relative expression on the y axis and cluster identity on the x axis. Violin plots are presented to demonstrate relative expression of the marker compared to other clusters. On the violin plots, each dot represents a single cell and the color is consistent with cluster color on the tSNE and UMAP plots (Figure 1a).

Identity
The 9 leading cell type markers identified on scRNAseq analysis for cluster 4 are presented. The data is presented with the relative expression on the y axis and cluster identity on the x axis. Violin plots are presented to demonstrate relative expression of the marker compared to other clusters. On the violin plots, each dot represents a single cell and the color is consistent with cluster color on the tSNE and UMAP plots (Figure 1 a).
Supplementary Figure 8: Conserved markers within human kidney cell cluster 5 The 9 leading cell type markers identified on scRNAseq analysis for cluster 5 are presented. The data is presented with the relative expression on the y axis and cluster identity on the x axis. Violin plots are presented to demonstrate relative expression of the marker compared to other clusters. On the violin plots, each dot represents a single cell and the color is consistent with cluster color on the tSNE and UMAP plots (Figure 1 a).
The 9 leading cell type markers identified on scRNAseq analysis for cluster 10 are presented. The data is presented with the relative expression on the y axis and cluster identity on the x axis. Violin plots are presented to demonstrate relative expression of the marker compared to other clusters. On the violin plots, each dot represents a single cell and the color is consistent with cluster color on the tSNE and UMAP plots (Figure 1 a).
RNASE7 mRNA expression in ICs vs non-ICs magnetically enriched from normal margins following kidney mass resection. RNASE7 was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for this comparison. Scatter plots with superimposed bar graphs are presented. RNASE7 is only present in ICs from 1 patient (biopsy 4) and absent in non-ICs from all patients. Each dot represents a RT-PCR run in a duplicate well N= 4 kidney tissue from distinct individuals. Source data are provided as a Source Data File. ND = not detected.
Supplementary Figure 18: RNASE7 expression is intermittently present in c-KIT+ (ICs) but not in c-KIT-(non-ICs) Supplementary Figure 19: Some collecting cell types have no changes in leading biological pathways with saline vs UPEC exposure Ingenuity™ pathway analysis results for human collecting duct cell types without pathway changes in UPEC exposed compared to saline control exposed cells. Top 10 pathways in NonA-nonB ICs/cluster 1 (C1) (a), Hybrid PC-IC/cluster 7 (C7) (b), PCs/cluster 10 (C10) (c) and B-ICs/cluster 11 (d) did not have differences in the order of the top 10 biological pathways as ranked by -log(p-value). A threshold (orange vertical line) of less than 1.3 -log(p-value) was used to assign significance. Data were analyzed through the use of IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis). IPA reports a p-value using a righttailed Fisher's exact test without multiple comparisons. However, the IPA input data includes the log2 fold change and an adjusted p-value from the Seurat scRNAseq analysis. Murine kidney cell suspension from "IC-reporter" mice was prepared. Cells were analyzed after acquisition by flow cytometry into a CD45 + fraction representing immune cells, red fluorescent protein variant tdTomato (tdT + ) fraction representing ICs and a CD45 -tdTfraction representing the remainder of the kidney cells and exposed to media alone or media containing a pH sensitive pHrodo Green E. coli BioParticles that cause cells to exhibit increased green fluorescence if bioparticles are phagocytized and acid is secreted into the phagosome. (a) In cells exposed to media alone, background green fluorescence uptake by cells was 0.7%, 0.0% and 0.0% of the CD45 + cells, tdT + ICs and tdTnon-IC kidney cells. (b) cells exposed to pHrodo Green E. coli BioParticles, 62.8% of CD45 + cells, 22.2% of tdT + ICs and 5.6% tdTnon-IC kidney cells expressed green fluorescence. The increase in the proportion of green fluorescence positive cells was higher in tdT + ICs compared to tdTnon-IC kidney cells following exposure to E. coli coated bioparticles, p <10E-15. The increase in proportion of green fluorescence positive cells was significantly higher in CD45 + cells than the tdT + ICs or tdTnon-IC kidney cells.
Proportions were analyzed with a 2-tailed Fisher Exact test. The data was obtained from 4 mouse kidneys pooled from 2 mice and run in a single experiment. Source data are provided as a Source Data File.
Supplementary Figure 21: The gating strategy to enrich ICs from mice (a) The gating strategy to show the enrichment of murine kidney ICs from saline and UPEC treated mice by flowsorting from V-ATPase-cre+tdTomato+ (IC reporter) mice. CD45-tdTomato+ cells (IC) were collected and used to detect Atp6v1b1 mRNA expression reported in Figure 6g. (b) The gating strategy to analyze uptake of pHrodo Green E. coli BioParticles gating strategy to analyze pHrodo Green E. coli BioParticle uptake by CD45+, CD45-tdTomato+ (IC) and CD45-tdTomato-(NIC) cells. Kidney cell suspension from V-ATPase-cre+tdTomato+ (IC reporter) mice was analyzed for pHrodo Green E. coli BioParticle particle uptake in the gated cells. This strategy was used for Supplementary Figure 20.
Gating strategy to analyze uptake of pHrodo bioparticles Gating strategy for flowsorting of murine ICs Supplementary Participants were aged 36-77 years. Ethnicity was "European-American" for four patients and "other" for one patient.
Supplementary Cluster 7 specific marker ^ "top 9" conserved genes in clusters presented in Supplemental Tables 3-14 Supplementary  Differential uptake of E. coli coated bioparticles by ICs in vivo. The linear regression results for each analyzed cell following intratubular injection of pHrodo Green E. coli BioParticles followed by intravital imaging and fluorescent quantification of GFP expression over is presented in table form. The slope representing the rate of increase in green fluorescence along with the R square , standard deviation of the values around the regression line (Sy.X), F distribution, degrees of freedom for the numerator and denominator (DFn and Dfd, p-values, whether the slopes differ significantly from a zero slope line and the number of values in each regression line are presented. Source data are provided as a Source Data File.