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Abstract: SA-PO291

Single-Cell Transcriptomics of Enriched Human Intercalated Cells Exposed to Uropathogen Reveal Differential Innate Immune Signature in Intercalated Cell Subsets

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Saxena, Vijay, Indiana University, Indianapolis, Indiana, United States
  • Schwaderer, Andrew L., Indiana University, Indianapolis, Indiana, United States
  • Hains, David S., Indiana University, Indianapolis, Indiana, United States
Background

We have previously shown that intercalated cells (ICs) are important for innate immunity in murine model of urinary tract infection. Little is known about their role in human innate immune function. We explored the whole genome transcriptome after early 1 hour uropathogen vs saline exposure in enriched human intercalated cell at single cell level.

Methods

Human ICs were enriched from kidney biopsy samples using magnetic cell sorting with anti-human c-kit microbeads after removal of dead cells and CD45+ immune cells. Enriched viable ICs were exposed to saline or UPEC for 1hr. Single cells were separated on 10x single cell instrument. Single cell gel beads containing barcoded oligonucleotides and reverse transcriptase reagents were generated with the v3 single cell reagent kit. Following cell capture and cell lysis, cDNA was synthesized and amplified. Illumina sequencing library was then prepared with the amplified cDNA. The resulting library was sequenced using Illumina NovaSeq 6000. 26 bp of cell barcode and UMI sequences and 91 bp RNA reads were generated. CellRanger 3.0.2 was utilized to process the raw sequence data generated. The R package Seurat development version 3.0.0.9 was used for the further gene expression analysis.

Results

Magnetically enriched CKIT+ cells expressed higher levels of V-ATPase mRNA expression compared to CKIT- cells. 6 clusters of collecting duct cells identified including 4 alpha IC clusters with variable SLC4A1 (AE1) and innate gene including DEFB1 (anti-microbial peptide) expression, 1 beta IC cluster (showing no innate immune gene expression) and 2 PC vs transitional IC/PC clusters (AQP2lo/hi V-ATPaselo) with distinct innate immune profile. Reactome pathway analysis predicted innate immune role for human ICs. Differential gene expression profiling found significantly upregulated gene with short UPEC exposure in IC clusters included Immunoglobulin lambda constant 3 (IGLC3) and adrenomedullin (ADM), an anti-microbial gene, integrin alpha E (ITGAE).

Conclusion

Innate immune function identified in murine models are conserved in human ICs. Enriched live human ICs can act as model system that can be used to determine human relevance of mouse findings. Single cell analysis reveals collecting cell types/subtypes are more diverse than previously recognized.

Funding

  • NIDDK Support