Background & Aims

The urinary bladder’s function depends on the intricate network of sensory neurons that innervate the bladder wall. Under normal conditions, this system allows for voluntary voiding without discomfort [2,3]. However, pathologies such as overactive bladder syndrome (OAB) and interstitial cystitis/bladder pain syndrome (IC/BPS) disrupt this harmonious process, leading to bladder pain and uncontrollable urgency [1,2]. The current medical care often fails to treat these conditions. This is a consequence of a critical knowledge gap in our understanding of the sensory neurons’ molecular mechanisms and their control of the bladder [2,3]. The aim of this study is to perform a comprehensive transcriptomic analysis of sensory neurons that innervate the bladder under both healthy and inflamed states. By characterizing the molecular changes that occur in sensory neurons during bladder inflammation, we hope to illuminate the pathophysiological processes that may contribute to OAB and IC/BPS.

Methods

Unbiased single-cell RNA-sequencing was performed on retrograde labeled mouse bladder sensory neurons isolated from lumbosacral dorsal root ganglia. We injected the fluorescent label Wheat Germ Agglutinin-conjugated (WGA) Alexa Fluor 647 into the bladder wall 7 days prior to DRG isolation, thus allowing specific identification of bladder-innervating neurons. Bladder inflammation was induced by intraperitoneal injection of 300 mg/kg cyclophosphamide. Control animals were injected with vehicle. After 24 h of cyclophosphamide/vehicle injections, L5, L6, and S1 DRGs were harvested and dissociated. Individually labeled neurons were collected from primary cultures, and single-cell RNA-seq (Smart-seq2) was performed (control x inflamed bladder). Some of the identified up/downregulated genes resulting from transcriptomics data were selected and validated by performing single-molecule fluorescent RNA in situ hybridization (RNAscope) in intact DRG sections.

Results

Transcriptomic analysis revealed approximately 1350 genes differentially expressed in bladder inflammation. Pathway enrichment analysis (Kegg annotation) showed multiple enriched pathways, such as bladder cancer, cholinergic/dopaminergic/GABAergic synapse, apoptosis, chemokine signaling, among others. We were also able to identify upregulated genes that are generally involved in nociceptive signal transduction: TRP channels, voltage-gated Ca/Na channels, cannabinoid receptors, Gaba receptors, plus many other targets less known in bladder disorders. We selected five genes for RNAscope validation of the single-cell sequencing data. All of them reflected the smart-seq data: bladder inflammation-induced upregulation of a selected transcription factor, superoxide dismutase enzyme, chloride and calcium channels. On the other hand, we demonstrated mRNA downregulation a Kv channel-interacting protein (using both methods).

Conclusions

This research marks an advancement in the understanding of bladder physiology, providing a detailed transcriptomic profile of sensory neurons innervating the bladder wall. The elucidation of gene expression changes offers a great advancement on how inflammation affects neuronal function, laying the foundation for a deeper characterization of these sensory neurons. The validation of the method through RNAscope strengthens the credibility of our extensive dataset. Looking ahead, we aim to translate our transcriptomic findings to functional assays, specifically through electrophysiology experiments comparing responses from neurons innervating healthy x inflamed bladder. This will hopefully cement the connection between genetic modulation and neuronal behavior in health and disease. The data generated in this research adds a valuable layer to the existing knowledge of bladder neurophysiology,and it may shed light on new therapeutic approaches for bladder pain syndrome and other conditions.

References

[1]Vanneste M, Mulier M, Nogueira Freitas AC, Van Ranst N, Kerstens A, Voets T, Everaerts W. TRPM3 Is Expressed in Afferent Bladder Neurons and Is Upregulated during Bladder Inflammation. Int J Mol Sci. 2021 Dec 22;23(1):107. doi: 10.3390/ijms23010107. PMID: 35008533; PMCID: PMC8745475.
[2]Grundy L, Wyndaele JJ, Hashitani H, Vahabi B, Wein A, Abrams P, Chakrabarty B, Fry CH. How does the lower urinary tract contribute to bladder sensation? ICI-RS 2023. Neurourol Urodyn. 2023 Oct 30. doi: 10.1002/nau.25316. Epub ahead of print. PMID: 37902296.
[3]Vanneste M, Segal A, Voets T, Everaerts W. Transient receptor potential channels in sensory mechanisms of the lower urinary tract. Nat Rev Urol. 2021 Mar;18(3):139-159. doi: 10.1038/s41585-021-00428-6. Epub 2021 Feb 3. PMID: 33536636.

Presenting Author

Ana C N Freitas

Poster Authors

Ana C N Freitas

PhD

VIB-Ku Leuven

Lead Author

Marie Mulier

Lead Author

Ashlee Caldwell

Lead Author

Andrei Segal

Lead Author

Matthias Vanneste

Lead Author

Nele Van Ranst

Lead Author

Axelle Kerstens

Lead Author

Suresh Kumar Poovathingal

Lead Author

Wouter Everaerts

Lead Author

Thomas Voets

PhD

Laboratory of Ion Channel Research, KU Leuven; IB Center for Brain & Disease Research

Lead Author

Topics

  • Mechanisms: Biological-Molecular and Cell Biology