The Polycystic Kidney Disease Cyst Transcriptome Defined by Integrating Spatial, Single Nuclear, and Bulk Transcriptomics.

Abstract

Background: Autosomal dominant polycystic kidney disease (ADPKD) has been transcriptionally profiled at single cellular resolution, yet dissociation eliminated cyst-defining morphology to preclude a retrograde analysis of the cyst transcriptome. Transcriptional profiling of dissected ADPKD cysts has demonstrated size-based transcriptional heterogeneity, impossible to define in prior single cell analysis, but sampling was neither single cell nor comprehensive to limit downstream transcriptional analysis.

Methods: 10X Genomics Visium spatial transcriptomics of an autosomal dominant polycystic kidney disease patient sample morphologically recognizes cysts to spatially characterize their ligand-receptor engagement, the signaling pathways of cystic transformation, and candidate cystic anchor genes that are specific and sensitive for cystic epithelia. Anchor gene validation in single nuclear RNA sequencing and cyst bulk RNA sequencing enhanced resolution, extended to cysts of variable size, and corroborated across ADPKD samples, prior to confirmatory confocal microscopy for protein-level localization.

Results: The polycystic kidney disease interactome may be enriched for vscular endothelial growth inhibitor-mediated hypoxia, fibrotic extracellular matrix, and proinflammatory transforming growth factor-β (TGF-β), tumor necrosis factor, and interferon-γ signaling. Enhanced ligand-receptor engagement between cysts and fibroblasts suggests Osteopontin and Macrophage migration inhibitory factor signals emanate from cysts and TWEAK, Tenascin-C, and Pleiotrophin signals into cysts. Cystic principal cells, standardized to non-cystic counterparts, implicates Hippo (YAP/TAZ), transforming growth factor-β (TGF-β/SMAD3), and mammalian target of rapamycin (mTORC2/SGK1) signaling in cystic transformation, while pathway analysis newly implicates nicotinic acetylcholine, cadherin, toll-like, and cholecystokinin receptor signaling. Top DEGs of cystic principal cells, interrogated for their specificity for cysts and their consistency across individual samples of single cell and cyst bulk RNA-seq, prioritizes musculin as a cystic anchor gene.

Conclusions: Spatial transcriptomics morphologically and transcriptionally characterizes the polycystic kidney disease transcriptome. Ligand-receptor, pathway, and reactome analyses may provide clinically-relevant disease associations that inform mechanistic studies in animal and organoid models. Integrating spatial, single nuclear, and cyst bulk RNA-seq spatially recognizes cysts at single cell resolution, while accounting for size-based heterogeneity of cysts.