Genomics and Histopathology in Interstitial Cystitis/Bladder Pain Syndrome
Hannah Ruetten and LaTasha K. Crawford contributed equally to this study.
ABSTRACT
Aims
In April of 2025, a Global Consensus meeting on IC/BPS was held in Winston-Salem, NC. The goal of this meeting was to establish global consensus in diagnostic criteria, phenotyping, treatment outcome assessment, and possible etiopathology in interstitial cystitis/bladder pain syndrome (IC/BPS). Our sub-committee focused on developing a consensus document on histopathology in IC/BPS.
Methods
Narrative review.
Results
Herein we discuss histological and molecular distinctions of Hunner lesion disease (HLD) and non-Hunner lesion disease (non-HLD) in IC/BPS, including urothelial alterations, inflammatory changes, vascularization and fibrosis, and neurophysiological dysfunction. The molecular and histological characteristics of HLD make it distinct from non-HLD. HLD is histologically characterized by urothelial denudation and subepithelial chronic inflammation featured by B-cell dominant lymphoplasmacytic infiltration, while non-HLD shows subtle inflammatory changes with preserved urothelial layers. Some cases of non-HLD reflect a component of multi-systemic pain syndrome driven by altered neurophysiological networks within the central or peripheral nervous system.
Conclusions
Molecular and histological characteristics revealed that HLD and non-HLD are distinct disease entities as the former is an inflammatory disease of the urinary bladder and the latter may be represented by systemic neurophysiological disorder, rather than pathology that is limited to the bladder. This concept could be useful in phenotyping, diagnosis, and development of biomarkers for IC/BPS.
Trial Registration
No new data were generated for this manuscript; no clinical trial was conducted.
1 Introduction
Interstitial cystitis/bladder pain syndrome (IC/BPS) is a symptom syndrome complex, clinically characterized by persistent bladder/pelvic pain during filling that is relieved by emptying, and lower urinary tract symptoms such as urinary frequency and urgency, in patients without identifiable bladder pathology including urinary tract infections and neoplasia [1]. Diagnosis of IC/BPS does not currently require tissue sampling and histological evaluation, however, past studies report histological features that can identify distinct patient subgroups or prognostic indicators for IC/BPS. There has also been a recent movement to include more specific evaluation criteria for IC/BPS bladder biopsy tissue to improve pathology report utility in patient diagnosis and treatment.
In April of 2025, a Global Consensus meeting on IC/BPS was held in Winston-Salem, NC. The goal of this meeting was to establish global consensus in diagnostic criteria, phenotyping and patient outcome assessment, and to discuss possible etiopathology for IC/BPS. Our sub-committee focused on developing a consensus document on histopathology in IC/BPS.
The IC/BPS patient population is heterogeneous and contains few well-defined subgroups of patients with unique disease phenotypes. One of the most widely accepted, clinically relevant subgroup distinctions is presence and absence of Hunner lesions. We will refer to patients in these subgroups as those with Hunner lesion disease (HLD) and those with non-Hunner lesion disease (non-HLD), respectively. Herein we discuss histological and molecular distinctions between HLD and non-HLD, including urothelial alterations, inflammatory characteristics, and neurophysiological dysfunction.
There are currently no standard guidelines for IC/BPS tissue sampling because bladder biopsy is not required for diagnosis. However, during cystoscopy, samples should be obtained when possible and sent to pathology for exclusion of neoplasia, infection, and other rare disorders such as eosinophilic cystitis. For diagnostic purposes, the most critical feature of sampling is not necessarily where the samples are obtained but instead that proper tissue handling and labeling has occurred. Care should be taken to avoid crush artifacts from forceps. If a Hunner lesion is sampled it should be placed in its own formalin container or in its own cassette and labeled as a Hunner lesion. If a normal area is collected this again should be labeled as “normal,” kept separate, and the location the biopsy was obtained from noted. For research purposes some groups obtain 2–3 cold-cup forcep biopsies from the posterior bladder wall, right above the trigone and place them directly into formalin or RNALaterTM. In research cases consistency is key so it is most important that similar samples are obtained from each patient and archived in similar media. In general tissue sampling technique is of high importance as the method of tissue acquisition and quality of tissue samples impacts all studies of genomics and histopathology.
2 Overview of HLD and Non-HLD
HLD is diagnosed based on the presence of the Hunner lesions on cystoscopy and also referred to as “Hunner-type IC,” “classic IC/BPS,” “ulcerative IC/BPS,” or “ESSIC criteria type 3.” Hunner lesions are characteristic reddish, hyperemic flat mucosal regions of the bladder, frequently accompanied by small vessels that radiate from the center of the lesions [2]. At the first endoscopic surgery in patients with HLD, Hunner lesions should be biopsied to rule out infectious or neoplastic disease. Histologically, Hunner lesions are characterized by (1) urothelial denudation, (2) dense inflammatory infiltrates predominantly composed of lymphocytes and plasma cells frequently accompanied by lymphoid follicles/aggregates, (3) subepithelial hemorrhage, neovascularization, fibrosis, or edema, and/or (4) subepithelial/detrusor mastocytosis or fibrosis [3-7].
Non-HLD lacks Hunner lesions on cystoscopy and corresponds to “ESSIC criteria type 1 and type 2.” Most of the non-HLD bladders retain full-thickness urothelial layers and show little inflammatory changes. Non-HLD is thought to be due to distinct, potentially neurophysiological etiopathogenesis, and it is more frequently associated with comorbid somatic pain, such as fibromyalgia and irritable bowel syndrome [3].
3 Urothelial Alterations
The luminal surface of the urinary bladder is a unique barrier comprised of urothelial cells bound by tight and adherens junctions. A glycocalyx of membrane bound uroplakins, lectins, and glycosaminoglycan (GAGs) overlies the most superficial cells of the urothelium. Under healthy conditions, the basal layer of GAG is composed of both chondroitin sulfate (CS) and heparan sulfate, while the superficial layer (luminal surface) is comprised primarily of CS [8]. The GAG layer, and CS in particular, are lost with disruption of the urothelial barrier in HLD [8, 9].
In all patients with IC/BPS, the urothelial barrier is compromised leading to increased urothelial permeability [10]. In HLD, the most severe urothelial lesions are found within the Hunner lesions, characterized by urothelial denudation with subepithelial granulation tissue and dense inflammation. The rest of the bladder is also altered with urothelial thinning, irregular erosion or defects of the superficial layers [11, 12], as well as decreased urothelial turnover [13] and chronic inflammatory changes similar to those in the Hunner lesions [5]. The urothelium in bladders of patients with non-HLD demonstrates subtle partial erosion, and degeneration in the superficial urothelium is noted compared to patients with stress urinary incontinence [14] and healthy controls [12]. Studies suggest an increase in apoptotic activities of the urothelium in HLD [13, 15, 16] which may contribute to urothelial alterations. Increased apoptosis within the urothelium of non-HLD bladders has also been identified compared to healthy controls [13].
3.1 Ultrastructural Changes
Ultrastructural changes are detected through either scanning or transmission electron microscopy which allows visualization of cell ultrastructure including cytoskeletal components and cellular junctions on a nanometer level resolution. Ultrastructural changes in the urothelium of HLD bladders include surface defects, loss of umbrella cells, loss of uroplakin plaque, and loss of tight junctions between umbrella cells [17]. These urothelial defects have been shown to correlate with more severe patient symptoms [17].
3.2 Molecular Changes
Molecular changes (alterations in DNA, RNA, and/or proteins) in the urothelium of HLD bladders include a loss in the density of all uroplakins [18] while the urothelium in non-HLD demonstrates an increase in gene and protein expression of uroplakin III and the uroplakin III-Delta4 mRNA splice variant [19]. A different study reported an increase in uroplakin III in all IC/BPS patients, though it is unclear whether this reflected a consequence of prior treatments, or potential feedback response to injury [20]. In all IC/BPS, there is a decrease/loss of proteins that link adjacent urothelial cells including the adherent junction marker E-cadherin; a severe loss of E-cadherin in HLD and mild to moderate decrease in non-HLD [13]. A loss of the tight junction marker ZO-1 has also been described in prior studies, but those studies grouped together both IC/BPS subtypes [15, 16]. Other studies found no difference ZO-1 from controls in either IC/BPS subtype [13].
Collectively, these data suggest disruption of the urothelial barrier underlies the pathophysiology of HLD and to a lesser extent non-HLD and may be associated with pain severity.
4 Inflammation
4.1 Inflammatory Characteristics
Patients with HLD show diffuse, mild to severe subepithelial inflammation characterized by lymphoplasmacytic infiltration [5, 21, 22], elevated levels of genes involved in T- and B-cell signaling pathways [23], and high expression of pro-inflammatory proteins [24]. Neutrophil and eosinophilic infiltration can be observed to some degree, but the number of infiltrating granulocytes is outnumbered by that of lymphoplasmacytic cells in the HLD bladder. Meanwhile, only 5%–20% of patients with non-HLD have subepithelial inflammation [5, 21]. Other studies have shown that up to 84% of non-HLD patient biopsies have moderate inflammation that is more prevalent in comparison to healthy controls [12], though non-HLD inflammation is considered less severe than HLD.
A prominent feature of HLD bladders are frequent lymphoid follicles/aggregates and abundant plasma cell infiltration [5]. These plasma cells show co-expression of the chemokine CXCR3 receptor, suggesting activated local inflammatory responses in the HLD bladder [25]. Other hallmark features of HLD bladder include the frequent clonal expansion of infiltrating B-cells [5]. Recent study suggests that a proliferation-inducing ligand (APRIL) and B-cell activating factor (BAFF) are potential regulators of B-cell clonal expansion in HLD [26, 27].
HLD bladder also shows high expression of interleukin 17 A (IL-17A) [28, 29]. IL-17A is a proinflammatory cytokine that is important in Th1/17 polarized immune responses and is prominent in a number of autoimmune diseases [28]. In further support of an autoimmune nature of HLD, a genome-wide association study (GWAS) identified risk loci in the major histocompatibility complex (MHC) region including a locus encoding amino acids within the antigen-peptide binding groove. This suggests that altered antigen presentation of MHC class II molecules may underlie the HLD pathophysiology [30]. An additional genome-based expression profiling study found upregulated MHC II molecules as well as leukocyte immunoglobulin-like receptors in the urothelium from patients with HLD [31].
When comparing the Hunner lesion areas to the background non-Hunner lesion areas in a patient with HLD, increased expression of transcription factors and cytokines, HIF1-alpha, IFN-gamma, and IL-2 were observed within the Hunner lesions despite similar numbers of infiltrating inflammatory cells throughout the entire HLD bladder [32]. HIF1-alpha is an important transcription factor involved in various biological processes, regulates cytokine production, and is activated in response to low oxygen levels. This evidence suggests that increased HIF1-alpha expression in Hunner lesions may reflect locally elevated tissue hypoxia which might be potentially responsible for the etiology of the lesions [32].
4.2 Nitric Oxide Synthase
Nitric oxide synthase (NOS) plays an important role in various functions of the body including regulation of blood flow, coagulation, innate immunity, and neurotransmission [33]. Elevated levels of NOS expression in patients with HLD has been reported [22], potentially resulting from chronic inflammatory responses. In patients with HLD, intensified inducible-NOS (iNOS) immunoreactivity is observed in both the urothelial layers and subepithelial inflammatory infiltrates [34]. Inducible-NOS (iNOS), which is formed in response to lipopolysaccharides and cytokines, is generally thought to be “harmful” NOS and inhibition of this enzyme may be beneficial in states of inflammation or injury [35].
4.3 Mast Cells
The role of mast cells in IC/BPS has been a highly debated topic. Most discrepancies in past studies could be associated with differences in methodology: staining methods used to identify mast cells, region of interest area (i.e., assessed biopsy), and control group selection. Some studies reported that HLD bladders have greater mast cell infiltration compared to non-HLD and non-inflamed control bladder tissues [14, 36, 37], while other studies found the reverse results [13]. Interestingly, mast cell numbers in the Hunner lesions were comparable to those in the bladder tissue of other chronic cystitis conditions with similar levels of inflammation [38], suggesting that mast cell density mirrors the degree of inflammation rather than types of disease.
5 Vascularization and Fibrosis
Vascular endothelial growth factor (VEGF) and CD31 are often assessed in immunohistochemistry to explore neovascularization and micro-vessel density. A study found that VEGF is upregulated in HLD bladder, predominantly detected in subepithelial mononuclear cells [27]. Another found that VEGF and CD31 are increased in all IC/BPS, compared to controls [37]. In addition, levels of VEGF and CD31 expression are strongly correlated with the O'Leary and Sant symptom index, O'Leary and Sant's Problem Index, and pain scores, suggesting a potential role of increased vascularization in pain generation [37].
A study found that upregulation of transforming growth factor-beta (TGF-beta), and increased fibrosis can be observed in both HLD and non-HLD bladders compared to non-IC/BPS controls [37]. The non-HLD bladders are reported to have more severe fibrosis, and increased expression of genes involved in pro-fibrotic pathways including TGBF-beta 1-3, SMAD2, SNAI 1-3, and TWIST [14]. The non-HLD bladders also have higher WNT11 expression than HLD or controls, which was shown to cause increased collagen production in cell culture studies [39].
6 Neural Dysfunction
Several studies have identified increased innervation in the subepithelial layer of IC/BPS bladders [12, 40-42]. Some aspects of this hyperinnervation could be due to autonomic motor neurons, as increased tyrosine hydroxylase (TH) is identified in the subepithelial layer (especially in the detrusor muscle and around the vessels) in both HLD and non-HLD bladders [43]—HLD bladders show more severe hyperinnervation [43] TH was previously thought to correspond solely to sympathetic motor innervation; however, recent evidence suggests TH is also expressed in bladder sensory neurons in mice [44].
Several studies have reported a link between substance P, sensory neurons, and mast cell responses. In addition to its role in vasodilation and vascular permeability, substance P, which is released from sensory nerve terminals, can drive mast cell migration and degranulation in skin [45, 46]. The role of substance P in bladder afferent neurons may be similar, as mast cell counts and tissue histamine levels are associated with innervation density in the HLD bladders [40], in conjunction with the elevated levels of mRNA of the Substance P receptor NK1 [47].
Expression of neurotrophic, proinflammatory, and pro-nociceptive mediators in IC/BPS bladder can drive neuronal excitability, growth of nerve terminals, and increase responses to neurotransmitters released from sensory neuron axon terminals. In both Hunner lesion areas and non-Hunner lesion areas of HLD bladder, increased gene expression of NGF, TRPM2, TRPV1, CXCL9 and decreased gene expression levels of TRPV4 are reported [18]. Non-Hunner lesion areas of HLD bladder also show increased gene expression of TRPM8, TRPV2, ASIC, and NGF [18]. Meanwhile, the non-HLD bladders show increased gene expression levels of NGF, NTRK1, TRPV2, CCL21, and CXCL1 [18, 42].
Many studies in animal models suggest that primary afferent hyperexcitability and central nervous sensitization contribute to altered sensation and nociceptive responses in the bladder [3, 48]. Some cases of non-HLD may reflect a component of multi-systemic pain syndrome driven by altered neurophysiological networks within the central nervous system, which result in neural plasticity and central sensitization [49, 50]. Peripheral sensitization remains poorly understood in patients with IC/BPS, and further studies on the role of bladder afferent neural plasticity in IC/BPS symptoms are needed. Advances in neuro-urology research could provide key insights that further our understanding of IC/BPS etiology and aid in the development of clinically relevant research endpoints and biomarkers.
Author Contributions
Yoshiyuki Akiyama developed an outline for the manuscript, Yoshiyuki Akiyama organized meetings between authors, Hannah Ruetten and LaTasha K. Crawford drafted manuscript; Hannah Ruetten, LaTasha K. Crawford, Elise J. B. De, Wencheng Li, and Yoshiyuki Akiyama edited and revised manuscript; Hannah Ruetten, LaTasha K. Crawford, Elise J. B. De, Wencheng Li, and Yoshiyuki Akiyama approved final version of manuscript.
Acknowledgments
The authors would like to thank Gopal Badlani and Steve Walker for organizing the Global Consensus on IC/BPS, therein providing an opportunity for IC/BPS researchers to gather and stimulate discussions that will progress the IC/BPS research field.
Ethics Statement
The authors have nothing to report.
Consent
The authors have nothing to report.
Conflicts of Interest
The authors declare no conflicts of interest.
Open Research
Data Availability Statement
The authors have nothing to report.
