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Abstract: FR-PO146

scRNA-Seq Analysis of Polyploid Tubular Epithelial Cells Reveals a Specific Hypertrophy Program Triggered in Response to AKI

Session Information

  • AKI: Mechanisms - II
    November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Authors

  • De Chiara, Letizia, Universita degli Studi di Firenze, Firenze, Toscana, Italy
  • Lazzeri, Elena, Universita degli Studi di Firenze, Firenze, Toscana, Italy
  • Romagnani, Paola, Universita degli Studi di Firenze, Firenze, Toscana, Italy
Background

Acute Kidney Injury (AKI) is characterized by a rapid deterioration of kidney function. Recently, we showed that tubular epithelial cells (TC) respond to AKI by triggering polyploidization mediated hypertrophy, (i.e. increase their DNA content and dimension) which allows for a quick recovery of kidney function by providing increased functional output via cell hypertrophy. However, the processes that govern this response are currently unknown. Moreover, in the kidney polyploid TC mostly remain mononuclear, making their recognition challenging unless multiple targeted techniques are combined. Here, we explored the possibility to use single cell RNA-sequencing (scRNA-seq).

Methods

To identify polyploid TC, study their phenotype and dissect the mechanisms underlying polyploidization mediated hypertrophy, we applied scRNA-seq analysis in vitro and in vivo after ischemic injury combined with specific knocked-down experiments, cell sorting, pharmacological inhibition, chromatin-immunoprecipitation assay (ChiP) and in vivo transgenic models.

Results

Based on combined measurement of DNA content and cell cycle phase with the FUCCI reporter, we showed that primary proximal tubular cells (PTC) contain a fraction of polyploid TC. scRNA-seq of PTC revealed the presence of genes previously associated with different phases of polyploidization-mediated hypertrophy. A trajectory analysis suggested that PTC polyploidization starts with increased ribosome biogenesis, culminating in a YAP1 enriched signature. Sorting of polyploid PTC confirmed the YAP1 signature enrichment. scRNA-seq analysis on mouse kidneys at days 0, 2 and 30 after AKI confirmed our in vitro observations and suggested that polyploid TC have significantly increased transcriptome abundance, consistent with their increased DNA content. 2 days after AKI, TC specific YAP1 knock-out mice displayed less polyploid cells, smaller kidneys and TC compared to wild-type, in line with the concept that polyploid cells are hypertrophic. Finally, ChiP assays combined with targeted silencing confirmed YAP1 as the primary driver of TC polyploidization.

Conclusion

In conclusion: 1) scRNAseq can be successfully applied to study polyploidization if combined with the right tools and 2) scRNAseq revealed the presence of a specific hypertrophy program in polyploid TC driven by YAP1 activation.