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Single cell RNA-seq reveals the urothelium and stroma cell heterogeneity in acute and chronic injured urinary bladder by single-cell analysis

ABSTRACT: Some studies have demonstrated the heterogeneity of bladder epithelium and fibroblasts, as well as their crosstalk during bladder development and regeneration. However, due to the limitations of techniques, a comprehensive understanding of cellular heterogeneity and crosstalk during bladder regeneration is still lacking. Here, we used droplet-based scRNA-seq to recover the heterogeneity of urothelial cells, cell cycle characteristic of proliferative cell population and proliferative niches after acute and chronic injury induced by cyclophosphamide.

ORGANISM(S): Mus musculus

PROVIDER: GSE164557 | GEO | 2021/05/12

REPOSITORIES: GEO

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Project description:The kidney and bladder expresses a variety of powerful defense mechanisms to limit urinary infection including iron scavenging, a process called "nutritional immunity". We previously demonstrated that bladder and kidney epithelia generates the archetype of the urinary iron defense, a lipocalin called NGAL which binds Enterochelin, a bacterial siderophore. However, because urinary bacteria can evade the bacteriostatic effects of NGAL by modifying Enterochelin and by producing additional siderophores, other mechanisms of "nutritional immunity" are anticipated. To study the defense in the kidney, we used a cell type specific method of RNA isolation in mouse (adapted from Gay et al, Oregon). A promiscuous form of phosphoribosyltransferase (UPRT) was cloned into the Rosa26 locus using a floxed-stop design which we activated in a cell specific fashion with Atp6v1b1-Cre. Thiouracil was introduced 12hr and 24hrs after urinary tract infection. Isolation of RNA directly from intercalated revealed synthetic (Npas1-Bmal1-Alas), transport (Hrg1, Flvcr1) and metabolic enzymes (Hmox1) of heme metabolism. Hmox1 was detected in the kidney with a luciferase based reporter (Contag et al, Stanford) after infection. CO production was detected in vivo and in AtpCre-mTmG FACS isolated intercalated cells in vitro using a novel metal-based probe synthesized at Columbia (adapted from Liu et al, China). Urinary bacteria (UPEC) with heme transport mutations were not competitive in the colonization of the kidney but conversely were markedly stimulated by iron, suggesting bacterial-host competition for heme capture and metabolism. In fact, infection upregulated both heme synthetic and metabolic genes, suggesting that CO production is induced by infection. Exposure to CO terminated the growth of UPEC. In sum, we have identified an unusual iron trafficking system in the collecting ducts that mirrors the nutritional requirements of UPEC to achieve pyelonephritis. Many of these components are specific to the intercalated cells, consistent with the notion that these cells defend the urinary tract from infection.

Project description:To date, there are no efficacious translational solutions for end-stage urinary bladder dysfunction. Current surgical strategies including urinary diversion and bladder augmentation enterocystoplasty (BAE) utilize autologous intestinal segments (e.g. ileum) to increase bladder capacity to protect renal function. Considered the standard of care, BAE is fraught with numerous short- and long-term clinical complications. Previous clinical trials employing tissue engineering approaches for bladder tissue regeneration have also been unable to translate bench-top findings into clinical practice. Major obstacles still persist which need to be overcome in order to advance tissue engineered products into the clinical arena. These include scaffold/bladder incongruencies, the acquisition and utlity of appropriate cells for anatomic and physiologic tissue recapitulation, and the choice of appropriate animal model for testing. Here we demonstrate that the elastomeric, bladder biomechanocompatible poly(1,8-octamethylene-citrate-co-octanol) (PRS; synthetic) scaffold co-seeded with autologous bone marrow-derived mesenchymal stem cells (MSCs) and CD34+ hematopoietic stem/progenitor cells (HSPCs) supports robust long-term, functional bladder tissue regeneration within the context of a clinically relevant baboon bladder augmentation model simulating bladder trauma. Partially cystectomized baboons were independently augmented with either autologous ileum or stem cell-seeded small intestinal submucosa (SIS; a commercially available biological scaffold) or PRS grafts. Stem cell synergism promoted functional tri-layer bladder tissue regeneration including whole graft neurovascularization in both cell-seeded grafts. However, PRS-augmented animals demonstrated fewer clinical complications and more advantageous tissue characterization metrics compared to ileum and SIS-augmented animals. Two-year study data demonstrate that PRS/stem cell-seeded grafts drive bladder tissue regeneration and are a suitable alternative to BAE.

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Single cell RNA-seq reveals the urothelium and stroma cell heterogeneity in acute and chronic injured urinary bladder by single-cell analysis

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