The Use of SPIM to Target Podocyte Cells in the Zebrafish Kidney

Abstract

Damaged podocyte cells are a common pathology in a myriad of renal diseases. Elucidating the mechanisms behind the occurrence of damage within podocytes is vital to improving our ability to mitigate, prevent and contribute to new therapies for treating kidney conditions. The zebrafish provides an ideal model to visualise podocytes in vivo, and we have established tg(Pod:GCaMP6s-LifeactTagRFP-T) transgenic fish expressing the fluorescent reporters GCaMP6s and LifeAct-red fluorescent protein (RFP) under the control of the podocin promotor. We have used selective plane illumination microscopy (SPIM) to capture high-resolution images of podocytes within live zebrafish larvae and to induce controlled podocyte damage. Expression of GCaMP6s enabled us to visualise changes in calcium ion dynamics, a known response to podocyte damage, and undertake detailed pathological analyses via electron microscopy to assess changes in cell and tissue morphology. To provide a platform for future comparative studies of kidney injury, we used FACs to isolate podocytes from tg(Pod:GCaMP6s-LifeactTagRFP-T) fish for the generation of a single cell RNA sequencing (scRNAseq) library for sequencing and bioinformatics analysis. The scRNAseq data from podocytes shows distinct populations of cells and potential gene switching between clusters, and we identified podocyte transcripts encoding proteins related to calcium-binding and actin filament assembly in common with those expressed in human and mouse mature podocytes.