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| Status |
Public on Oct 22, 2020 |
| Title |
Kidney Intercalated Cells Phagocytose and Acidify Escherichia coli |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Intercalated cells are known to be involved in acid-base homeostasis via vacuolar ATPase (H+-ATPase or V-ATPase) expression. Increasing evidence supports an innate immune role for ICs along with their traditional function of pH regulation. In this study, human kidney tissue was enriched for viable intercalated cells then exposed to uropathogenic E. coli versus saline control. Single cell transcriptomics was performed. Six intercalated cell subtypes were identified including hybrid principal-intercalated cells. Cell specific cluster marker gene list generated from this sequencing data was put through ingenuity pathway analysis pipeline which predicted “phagosome maturation” as a key biological pathway that increased in rank following exposure to uropathogenic E. coli in two of the intercalated cell subtypes. Uptake of E. coli and pHrodo coated E. coli BioParticlesTM during live animal intravital microscopy demonstrated that intercalated cell phagocytosis of bacteria was an active process that involved acidification. Taken together, our finding indicate that intercalated cells represent an epithelial cell with characteristics of professional phagocytes like macrophages or neutrophils, which includes the ability to phagocytose E. coli and acidify phagolysosomes.
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| Overall design |
Normal margin of the human kidney biopsy sample was processed for single cell suspension with enzymatic digestion (Liberase TL and DNAse I) and rapid dissociation using GentleMacs (Miltenyi Biotec). Dead cells were then removed from single cell suspension using dead cell removal microbead (Miltenyi Biotec). CD45+ traditional immune cells were then removed using anti-human CD45 microeads (Miltenyi Biotec). Intercalated cells were then enriched using anti-human C-KIT (CD117) microbeads (Miltenyi Biotec). Cell viability was tested on hemocytometer. Viable cells were equally divided into 2 wells of the 96-well U bottom plate and exposed to uropathogenic E.coli (UPEC) for 1 hr and sterile saline at 370C and 5% CO2 environment. After washing with sterile PBS and re-suspension in PBS (without Ca2+ and Mg2+) both cells samples (Saline control and UPEC exposed) were immediately processed for single cell sequencing using 10x genomics platform. Single cells of both samples were sequenced on Illumina Novaseq 6000 instrument.
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| Contributor(s) |
Saxena V, Gao H, Arregui S, Zollman A, Kamocka MM, Xuei X, McGuire P, Hutchens M, Hato T, Hains DS, Schwaderer AL |
| Citation(s) |
33893305 |
| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| R01 DK106286 |
The interface between critical acid-base mediators and the renal bacterial defense |
INDIANA UNIVERSITY |
Andrew Lawrence Schwaderer |
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| Submission date |
Oct 21, 2020 |
| Last update date |
May 07, 2021 |
| Contact name |
Andrew Schwaderer |
| E-mail(s) |
[email protected]
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| Organization name |
Indiana University School of Medicine
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| Department |
Pediatric Nephrology
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| Street address |
1044 West Walnut Street
|
| City |
Indianapolis |
| State/province |
IN |
| ZIP/Postal code |
46202-5254 |
| Country |
USA |
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| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (2) |
| GSM4847759 |
CKIT micorbead enriched and Saline exposed cells |
| GSM4847760 |
CKIT micorbead enriched and UPEC exposed cells |
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| Relations |
| BioProject |
PRJNA670499 |
| SRA |
SRP288044 |
