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Kidney Intercalated Cells Phagocytose and Acidify Escherichia coli

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE159805 | GEO | 2020/10/22

REPOSITORIES: GEO

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Project description:Purpose: Transport processes in the renal collecting duct are responsible for precise regulation of blood pressure and body fluid composition. The collecting duct is composed of at least three cell types, type A intercalated cells (A-IC), type B intercalated cells (B-IC) and principal cells (PC). To identify the genes that are selectively expressed in each cell type, including cell-surface receptors, transcription factors, transporters and secreted proteins, we used cell surface markers necessary for isolation of each of the three cell types using fluorescence-activated cell sorting and carried out single-cell RNA-Seq to measure the mRNA species in each of these cell types. Methods: We enriched each of the three cell types using fluorescence-activated cell sorting. Subsequently, we carried out single-cell RNA-Seq of those cells using a microfluidic chip (Fluidigm C1 system). Single-cell cDNA libraries were constructed for paired-end sequencing and sequenced on Illumina HiSeq3000 platform. In addition, we also microdissected mouse CCDs and cTALs and carried out single-tubule RNA-Seq. Reads were mapped to mouse Ensembl Genome by STAR and transcript abundances were calculated in the units of transcripts per million (TPM) using RSEM (https://github.com/deweylab/RSEM). Single-cell RNA-Seq data anaysis were carried out using Seurat package in R. Results and conclusion: Single-cell cDNA libraries were constructed for paired-end sequencing and at least 10-million sequence reads per cell were mapped to the mouse genome (Ensembl, GRCm38.p5). On average, cells were sequenced to a depth of 3000 genes (TPM>1). Unsupervised clustering analysis revealed five different cell types, namely type A intercalated cells (n=87), type B intercalated cells (n=23), principal cells (74), proximal tubule cells (n=19), and non-epithelial cells (n=32). The identified patterns of gene expression among A-ICs, B-ICs and PCs provide a foundation for understanding physiological regulation and pathophysiology in the renal collecting duct.

Project description:Analysis of epithelial explants injected with the intracellular domain of Notch (ICD) to block the formation of multi-ciliate and proton secreting cells or with dominant negative human Mastermind (HMM) to induce the formation of ectopic multi-ciliate and proton secreting cells. Results show which genes are up or down-regulated when HMM is compared to ICD. Specialized epithelial cells in the amphibian skin play important roles in ion transport, but how they arise developmentally is largely unknown. Here we show that proton-secreting cells (PSCs) differentiate in the X. laevis larval skin soon after gastrulation, based on the expression of “kidney” specific form of the H+v-ATPase that localizes to the plasma membrane, orthologs of the Cl-/HCO3- antiporters ae1 and pendrin, and two isoforms of carbonic anhydrase. Like PSCs in other species, we show that the expression of these genes is likely to be driven by an ortholog of foxi1, which is also sufficient to promote the formation of PSC precursors. Strikingly, the PSCs form in the skin as two distinct subtypes that resemble the alpha- and beta-intercalated cells of the kidney. The alpha-subtype expresses ae1 and localizes H+v-ATPases to the apical plasma membrane, whereas the beta-subtype expresses pendrin and localizes the H+v-ATPase cytosolically or basolaterally. These two subtypes are specified during early differentiation, and can be distinguished based on the expression of ubp1, a transcription factor in the grainyhead family. Finally, we show that ectopic expression ubp1 is sufficient to promote the differentiation of beta-subtypes while repressing alpha-subtypes. These results have implications for how PSCs are specified in vertebrates and become functionally heterogeneous. 2-cell stage Xenopus embryos were injected with synthetic mRNA encoding ICD or HMM. At stage 9/10 the presumptive ectoderm was cut off the embryo and cultured on a fibronectin coated coverslip. At stage 22 RNA was harvested from explants and used as the starting material for arrays.

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Kidney Intercalated Cells Phagocytose and Acidify Escherichia coli

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