Abstract: TH-OR018

Towards Single Cell RNA-Sequencing of Tubular Epithelium in AKI

Session Information

Category: Acute Kidney Injury

  • 001 AKI: Basic

Authors

  • Wu, Haojia, Division of Nephrology, Washington University in St. Louis, Saint Louis, Missouri, United States
  • Donnelly, Erinn L, Division of Nephrology, Washington University in St. Louis, Saint Louis, Missouri, United States
  • Morris, Samantha A, Department of Developmental Biology, Washington University in St Louis, Saint Louis, Missouri, United States
  • Humphreys, Benjamin D., Division of Nephrology, Washington University in St. Louis, Saint Louis, Missouri, United States
Background

A complete transcriptional atlas of epithelial states and dynamics during AKI and repair is a goal of the Kidney Precision Medicine Project. Here, we apply DropSeq, a microfluidic single cell RNA sequencing (scRNA-seq) technique, to characterize kidney tubule single cell transcriptional signatures. We additionally asked if MeOH fixation allows storage of single cells for subsequent scRNA-seq.

Methods

Mouse kidney was dissociated with Liberase TL and DNase I. Single cells were fixed by methanol and stored in -80°C for 4 days. Rehydrated cells were purified by FACS and DropSeq performed according to Macosko et al. Unsupervised clustering was performed to group the kidney cells into separate clusters based on the biological variations on gene expression. Cell types were annotated with known markers or by comparing to a published tubular cell transcriptional profiling dataset.

Results

High quality DropSeq cDNA libraries (average insert size of 1284bp) was generated from MeOH fixed kidney cells. We sequenced 3130 fixed cells at a depth of 7795 reads/cell, detecting an average of 2283 transcripts and 961 genes per cell. Unbiased clustering revealed 12 separate cell types in kidney. This included six tubular cell types, including proximal tubule (PT), Loop of Henle (LOH), distal tubule, connecting tubule and collecting duct. A majority of the cells (67.7%) expressed proximal tubular markers (Slc34a1 and Lrp2), highlighting the preference of PT cell type dissociation with the current protocol. Reclustering analysis of Slc34a1 expressing cells further revealed 5 separate subtypes within the PT cluster. These included three distinct PT subtypes that correspond to S1, S2 and S3 segments. Interestingly, we identified two distinct PT subtypes co-expressing LOH markers (e.g. Wfdc2 and Jun).

Conclusion

DropSeq can be performed on MeOH fixed mouse kidney cells. This will facilitate future analysis of human kidney, whose availability is unpredictable, and allow generation of biobanks for downstream scRNA-seq analysis. Our approach enriches for tubular cell types, including S1, S2 and S3 segments of the proximal tubule, making it well suited for analysis of acute tubular injury and repair at cellular resolution.

Funding

  • NIDDK Support