Introduction: Adult human kidneys contain a wide diversity of cell types, including podocyte, endothelial, tubule, and interstitial cells, all of which interact through complex signaling pathways. Human kidney organoids are a useful tool for studying kidney injury because they contain many cell types and can better model certain podocytopathies than 2D culture. Here, we injured vascularized human kidney organoids with doxorubicin (DR), a known glomerular nephrotoxin. We have found prior with qPCR that injured kidney organoid endothelial and podocyte populations possess an increase in apoptosis and inflammation markers and a decrease in cell health markers, such as cell adhesion markers. Immunofluorescence confirmed a decrease in endothelial cell number in injury as well. Prior studies have found that PCI-34051 (PCI), an HDAC8 inhibitor, ameliorates injury and fibrosis across multiple organ systems, however, its therapeutic mechanism in the kidney is unknown and it has not been studied in glomerular disease. We have found prior that injured kidney organoids treated with PCI have preserved cytoskeleton organization, downregulated apoptosis and vascular permeability pathways, and increased podocyte and endothelial cell health. In this study, we aimed to investigate specific cell-cell communication pathways involved in doxorubicin injury and PCI-mediated amelioration of these injuries with scRNAseq and ligand-receptor pair analysis among and between all cell types.
Materials and
Methods: Our group has generated vascularized kidney organoids using a novel method, which shows robust endothelialization, improvement in podocyte maturation and renin cell emergence. We performed single cell RNA sequencing (scRNAseq) at 3 days post injury on three conditions: (1) control uninjured, (2) DR-injured, and (3) DR-injured + concurrent PCI-treatment. We then analyzed cell-cell communication with the R package CellChat.
Results, Conclusions, and Discussions:
Results: Cell-cell communication analysis via CellChat revealed the complicated dynamics of pathways and specific ligand-receptor pairs in DR-injury and DR+PCI treatment conditions. We found a shift in cell types responsible for the majority of cell signaling from the control condition to the injury and PCI treatment conditions. Endothelial cells were responsible for most signaling in control organoids, but podocyte, tubule, and interstitial populations became more active in the DR and PCI conditions. CellChat analysis confirmed prior qPCR and immunofluorescence findings of upregulation of COL1A1 fibrosis pathway in all cell types of the DR-injured condition. DR also damaged endothelial cell-cell communication pathways involved in cell adhesion and polarization, such as PECAM1 and CDH1. We found FGF, NCAM, HSPG, and BMP pathways, which are described in literature to be potentially indicative of advanced renal repair, to be upregulated only in the DR+PCI condition. Finally, of critical relevance to glomerular health and disease is the communication between podocytes and endothelial cells through the VEGF pathway. We found the ligands and receptors of the VEGF pathway changed in injury and PCI treatment, where KDR and FLT1 became downregulated in injury, but in the DR+PCI condition, there was improvement in FLT1 signaling and presence of VEGFB and PGF.
Conclusions: Our findings support prior literature demonstrating DR as an effective model of kidney injury. Additionally, we corroborated an HDAC8 inhibitor, PCI-34051, as a potential therapeutic for induced kidney injury. We identified multiple repair and injury pathways, such as BMP, FGF, NCAM, and HSPG, upregulated in PCI treatment, as significant areas of future investigation. We also demonstrated advanced VEGF signaling in PCI-treatment.
Discussions: Taken together, this data emphasizes the injurious pattern of doxorubicin to podocytes and endothelial cells. Furthermore, we found that doxorubicin disrupted several critical cell-cell communication pathways between all cell types of the kidney organoid. We also investigated several pathways that have previously been identified as repair mechanisms and may be involved in PCI’s mechanism of recovery in the kidney. This work, in addition to future investigation in cell-cell interactions in injury and with HDAC8 inhibition, significantly advances the field to develop kidney injury therapeutics.
