Interstitial cystitis/bladder pain syndrome (IC/BPS) is often characterized as a condition primarily affecting the urothelium of the bladder which serves as a critical barrier against the passage of urine, solutes, and toxins across its cellular surface [1]. The predominant pathological features observed in IC/BPS bladders involve urothelial denudation and the presence of chronic inflammation. Although certain treatments, including intravesical instillation of hyaluronic acid and platelet-rich plasma injections, have been developed to target chronic inflammation, promote urothelial regeneration, and repair the urothelial barrier, their therapeutic efficacy remains limited due to the absence of crucial genes and signaling pathways associated with urothelial injury in HIC [2].
The urothelium is composed of a stratified epithelium consisting of three distinct cell layers: the superficial layer (umbrella cells), the intermediate cell layer, and the basal cell layer. Among the various pathological processes proposed in IC/BPS, barrier dysfunction stands out as of paramount importance. Nevertheless, the precise microstructural damage and the key signaling pathways responsible for this barrier dysfunction in the IC/BPS bladder remain elusive. Hunner-type IC/BPS (HIC) represents a severe phenotype, emphasizing the critical significance of investigating the mechanisms underlying urothelial damage in HIC. Such an investigation holds immense potential for advancing the treatment and prevention of all subtypes of IC/BPS.

To comprehensively elucidate the process of HIC urothelial destruction and shed light on the key molecular pathways governing urothelial barrier disruption, we harnessed the power of single-cell RNA sequencing—an exceptionally potent tool for unraveling the heterogeneity of a specific cell type within tissues. Subsequently, we constructed a comprehensive landscape of the HIC bladder and delineated the differentiation and developmental trajectory of HIC urothelium using Pseudotime analysis to precisely identify the injured cell types within the HIC urothelium. Furthermore, we rigorously substantiated the mechanism driving urothelial damage in HIC through a series of meticulously conducted in vitro and in vivo experiments. The research findings hold significant promise in identifying pivotal targets for the effective and functional regeneration of HIC urothelium and the restoration of the urothelial barrier. Moreover, the results potentially provide a robust theoretical foundation for tailoring individualized treatments for IC/BPS patients.

Through reclustering, we identified 8 distinct clusters of urothelial cells. There was a significant reduction in UPK3A+ umbrella cells and a simultaneous increase in progenitor-like pluripotent cells (PPCs) within the HIC bladder. Pseudotime analysis of the urothelial cells in the HIC bladder revealed that cells faced challenges in differentiating into UPK3A+ umbrella cells, while PPCs exhibited substantial proliferation to compensate for the loss of UPK3A+ umbrella cells. The urothelium in HIC remains unrepaired, despite the substantial proliferation of PPCs. Thus, we propose that inhibiting the pivotal signaling pathways responsible for the injury to UPK3A+ umbrella cells are paramount for restoring the urothelial barrier and alleviating lower urinary tract symptoms in HIC patients. Subsequently, we identified key molecular pathways (TLR3 and NR2F6) associated with injury of UPK3A+ umbrella cells in HIC urothelium. Finally, we conducted in vitro and in vivo experiments to confirm the potential of the TLR3-NR2F6 axis as a promising therapeutic target for HIC.

1.	UPK3A+ umbrella cell is the first cell type to be damaged in HIC urothelium.
2.	UPK3A+ umbrella cell damage mediated by TLR3-NR2F6 triggers programmed destruction of urothelium in HIC (Figure 1).

UPK3A+ umbrella cell damage mediated by TLR3-NR2F6 triggers programmed destruction of urothelium in ulcerative IC/BPS, suggesting the potential of the TLR3-NR2F6 axis as a promising therapeutic target for HIC.

Peng L, Jin X, Li BY, et al. Integrating single-cell RNA sequencing with spatial transcriptomics reveals immune landscape for interstitial cystitis. Signal Transduct Target Ther 2022; 7: 161.
Peng L, Li BY, Wang W, et al. Identification of key genes in human urothelial cells corresponding to interstitial cystitis/bladder pain syndrome in a lipopolysaccharide-induced cystitis model. Neurourol Urodyn 2021; 40: 1720-1729.

Continence 12S (2024) 101469
DOI: 10.1016/j.cont.2024.101469