Urothelial Repair Research Articles

Conditional Knockout of Glycosaminoglycan Synthesis in Bladder Epithelium Has No Effect on Urinary Voiding in Mice

Glycosaminoglycans (GAGs) on the luminal surface of the bladder epithelium have been suggested to play an important role in barrier function and in protection from infection. There is also a substantial literature that posits a breakdown in the GAG layer may underlie, or be associated with the development of painful bladder syndrome (PBS, aka interstitial cystitis) and indeed, GAG mimetics such as pentosan polysulphate provide symptomatic relief for some patients. However, opinions on the presence and role of GAGs vary, and incontrovertible evidence is lacking. We have taken a unique approach to answering this question by engineering a highly cell-specific genetic mouse model that lacks the obligatory initiating enzyme for GAG synthesis in urothelium. Uroplakin 2-Cre (UP2-Cre) mice were crossed with xylosyltransferase 2-floxed mice to knockout the xylt2 gene in urothelium. RT-PCR confirmed the tissue specificity of the knockout in urothelium but not in bladder smooth muscle. Further, lacZ reporter staining in cryosections demonstrated highly specific expression of the Cre construct in urothelium. Mammals express two xylosyltransferases, XylT1 and XylT2 in different proportions in different tissues. While functionally similar they appear to differ in their substrate specificity and kinetics. qPCR in dissected urothelium demonstrated that XylT2 was the dominant isoform, being expressed at approximately 80-fold higher level than XylT1. Void spot assays (VSA) on filter paper (4 hours) have proven to be a reliable indicator of voiding dysfunction in mice. We conducted VSAs on female Up2 cre/xylt2 fl/fl (n=12) and xylt2 fl/fl (n=11) littermate controls aged 8 – 16 weeks. There was no significant difference in void volumes, number of primary voids, volume/void or number of microvoids (<20 μl) between controls and knockouts, whereas transurethral instillation of protamine sulfate, which injures the apical cell layer, induced pain and a significantly greater number of microvoids, with reduced primary void volumes. We conclude preliminarily, that loss of GAGs in urothelium does not affect bladder function, suggesting that GAGs do not play a role in protecting the suburothelium and adjacent sensory afferents from noxious urine components. Future work will characterize and quantitate the distribution of GAGs within the urothelium to determine whether GAGs are deployed on the luminal surface and, whether they play a role in urothelial repair from injury. NIH grant P20DK097818. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Diabetes causes NLRP3-dependent barrier dysfunction in mice with detrusor overactivity but not underactivity.

Approximately half of the patients with diabetes develop diabetic bladder dysfunction (DBD). The initiation and progression of DBD is largely attributed to inflammation due to dysregulated glucose and the production of toxic metabolites that activate the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome. NLRP3 activation leads to the production and release of proinflammatory cytokines and causes urothelial pyroptosis, a form of programmed cell necrosis, which we hypothesize compromises urothelial barrier integrity. Here, we investigated how NLRP3-dependent inflammation impacts barrier function during the progression of diabetes using a type 1 diabetic female Akita mouse model that progresses from an early overactive to a late underactive detrusor phenotype at 15 and 30 wk, respectively. To determine the specific role of NLRP3, Akita mice were crossbred with mice lacking the NLRP3 gene. To determine barrier function, permeability to small molecules was assessed, ex vivo using Evans blue dye and in vivo using sulfo-NHS-biotin. Both ex vivo and in vivo permeabilities were increased in diabetic mice at 15 wk. Expression of uroplakin and tight junction components was also significantly downregulated at 15 wk. Interestingly, diabetic mice lacking the NLRP3 gene showed no evidence of barrier damage or downregulation of barrier genes and proteins. At the 30-wk time point, ex vivo and in vivo barrier damage as well as barrier component downregulation was no longer evident in diabetic mice, suggesting urothelial repair or remodeling occurs between the overactive and underactive stages of DBD. Collectively, these findings demonstrate the role of NLRP3-mediated inflammation in urothelial barrier damage associated with detrusor overactivity but not underactivity.NEW & NOTEWORTHY This is the first study to demonstrate that NLRP3-mediated inflammation is responsible for urothelial barrier damage in type 1 diabetic female Akita mice with an overactive bladder. Eliminating the NLRP3 gene in these diabetic mice prevented barrier damage as a result of diabetes. By the time female Akita mice develop an underactive phenotype, the urothelial barrier has been restored, suggesting that inflammation is a critical causative factor early in the development of diabetic bladder dysfunction.

Durability of and role of AKT in FGF7p urothelial protection against cyclophosphamide.

We previously identified a peptide derived from human fibroblast growth factor 7 (FGF7p) that blocks urothelial apoptosis similar to full‐length FGF7, although effects of FGF7p on urothelial repair are unknown. Also, while urothelial AKT activation downstream of FGF7p correlated with the anti‐apoptotic effects, we have not directly interrogated the role of AKT in mediating the cytoprotection. Our goal was to assess effects of FGF7p on urothelial repair and the role of AKT signaling in mediating the cytoprotective effects of FGF7p. We performed hematoxylin and eosin (H&E), TUNEL, and/or immunofluorescence (IF) staining for various markers in FGF7p‐treated mice 28 days after giving cyclophosphamide or after co‐administering a systemic AKT antagonist with FGF7p 24 h after cyclophosphamide. Vehicle‐treated and injured mice had hyperplastic urothelium, incomplete return of mature superficial cell markers, ongoing proliferation, and continued presence of basal progenitor markers 28 days after injury; conversely, FGF7p‐treated mice had normal numbers of urothelial cell layers, nearly complete return of superficial cell markers, limited proliferation and fewer basal progenitor cells 28 days post‐injury. Vehicle‐treated mice also had ectopic lumenal basal progenitor cell markers, while FGF7p had none 28 days after cyclophosphamide. Co‐administration of an AKT inhibitor largely abrogated FGF7p‐driven AKT activation and cytoprotection in urothelium 24 h after injury. Thus, FGF7p drives faster and higher fidelity urothelial repair by limiting apoptotic injury via AKT signaling, similar to full‐length FGF7. Finally, FGF7p is much less expensive to synthesize and has a longer shelf life and higher purity than FGF7.

Management of BK virus-associated haemorrhagic cystitis in allogeneic stem cell transplant recipients.

BK virus (BKV)-related haemorrhagic cystitis (HC) is an important cause of morbidity following allogeneic haematopoietic stem cell transplantation (HSCT). The various risk factors include high-level BKV viruria and/or viremia, myeloablative conditioning, acute graft versus host disease (GVHD), cytomegalovirus viremia, and unrelated or HLA-mismatched donor. The presence of high plasma BK viral load and cytopenias have been implicated as important predictors for protracted disease course. These patients frequently require hospitalisation which may extend for several weeks. Supportive measures in the form of analgesics, intravenous hydration, bladder irrigation, and transfusion support remain the mainstay of management. Various drugs have been used with limited success in this setting. These include antiviral drugs, fluoroquinolones, leflunomide, growth factors, clotting factors, estrogens, and prostaglandins. The role of adoptive cellular immunotherapy has also been explored but lacks clinical validation. The strategies aimed at expediting urothelial repair like hyperbaric oxygen therapy (HBOT), intravesical fibrin glue and platelet-rich plasma (PRP) are emerging. Some patients with severe disease do require surgical intervention to relieve urinary obstruction. The frequent co-occurrence of acute GVHD and CMV disease further complicates the management in such patients. There is an unmet need for effective and evidence-based options for the prevention and management of this disease. Due to lack of robust data supported by randomised trials, the acceptability of the available guidelines to simplify the treatment is expected to be low. Despite the availability of various treatment options, the management of BKV-related HC in day-to-day practice continues to be a challenge. The aim of this article is to put forward an up-to-date review of the preventive and therapeutic strategies for BKV-related HC.

Loss of Fibroblast Growth Factor Receptor 2 (FGFR2) Leads to Defective Bladder Urothelial Regeneration after Cyclophosphamide Injury

Cyclophosphamide may cause hemorrhagic cystitis and eventually bladder urothelial cancer. Genetic determinants for poor outcomes are unknown. We assessed actions of fibroblast growth factor receptor (FGFR) 2 in urothelium after cyclophosphamide exposure. Conditional urothelial deletion of Fgfr2 (Fgfr2KO) did not affect injury severity or proliferation of keratin 14+ (KRT14+) basal progenitors or other urothelial cells 1 day after cyclophosphamide exposure. Three days after cyclophosphamide exposure, Fgfr2KO urothelium had defective regeneration, fewer cells, larger basal cell bodies and nuclei, paradoxical increases in proliferation markers, and excessive replication stress versus controls. Fgfr2KO mice had evidence of pathologic basal cell endoreplication associated with absent phosphorylated ERK staining and decreased p53 expression versus controls. Mice with conditional deletion of Fgfr2 in urothelium enriched for KRT14+ cells reproduced Fgfr2KO abnormalities after cyclophosphamide exposure. Fgfr2KO urothelium had defects up to 6 months after injury versus controls, including larger basal cells and nuclei, more persistent basal and ectopic lumenal KRT14+ cells, and signs of metaplasia (attenuated E-cadherin staining). Mice missing one allele of Fgfr2 also had (less severe) regeneration defects and basal cell endoreplication 3 days after cyclophosphamide exposure versus controls. Thus, reduced FGFR2/ERK signaling apparently leads to abnormal urothelial repair after cyclophosphamide exposure from pathologic basal cell endoreplication. Patients with genetic variants in FGFR2 or its ligands may have increased risks of hemorrhagic cystitis or urothelial cancer from persistent and ectopic KRT14+ cells.

Platelet-Rich Plasma Ameliorates Cyclophosphamide-Induced Acute Interstitial Cystitis/Painful Bladder Syndrome in a Rat Model.

Background: Interstitial cystitis/painful bladder syndrome (IC/PBS) could be treated to ameliorate urothelial injury. Here, we investigated the efficacy of intravesical instillation with platelet-rich plasma (PRP) and hyaluronic acid for acute IC/PBS. Methods: The effects of PRP and hyaluronic acid on the proliferation of normal human fibroblast cells (HFCs) were assessed. Additionally, thirty virgin female rats were randomized into five groups: group 1, saline-injected control; group 2, cyclophosphamide (CYP) plus intravesical instillation with normal saline; group 3, CYP plus intravesical instillation with hyaluronic acid (1 mg/mL); group 4, CYP plus intravesical instillation with PRP; and group 5, CYP plus intravesical instillation with PRP plus hyaluronic acid. A cystometry and histological assessments were performed. The expression of cell junction-associated protein zonula occludens-2 (ZO-2) and inflammatory cytokine interleukin 6 (IL-6) was also measured. Results: Low dose PRP increased proliferation in HFCs. The acute IC/PBS rats showed significantly lower voiding interval values. Voiding interval values were significantly higher in the CYP plus intravesical instillation with PRP group than in the CYP-induced acute IC/PBS group. Additionally, the expression of ZO-2 was increased and IL-6 was decreased in the CYP plus intravesical instillation with PRP group compared with the CYP-induced acute IC/PBS group. Conclusion: These findings suggest that PRP modulate urothelial repair, which ameliorate the increase in urination frequency in rats treated with CYP. Overall, PRP may confer potential benefits by acting as urothelial repair modulators.

Polyploid Superficial Cells that Maintain the Urothelial Barrier Are Produced via Incomplete Cytokinesis and Endoreplication

SUMMARYThe urothelium is an epithelia barrier lined by a luminal layer of binucleated, octoploid, superficial cells. Superficial cells are critical for production and transport of uroplakins, a family of proteins that assemble into a waterproof crystalline plaque that helps protect against infection and toxic substances. Adult urothelium is nearly quiescent, but rapidly regenerates in response to injury. Yet the mechanism by which binucleated, polyploid, superficial cells are produced remains unclear. Here, we show that superficial cells are likely to be derived from a population of binucleated intermediate cells, which are produced from mononucleated intermediate cells via incomplete cytokinesis. We show that binucleated intermediate and superficial cells increase DNA content via endoreplication, passing through S phase without entering mitosis. The urothelium can be permanently damaged by repetitive or chronic injury or disease. Identification of the mechanism by which superficial cells are produced may be important for developing strategies for urothelial repair.

Therapeutic exploitation of IPSE, a urogenital parasite-derived host modulatory protein, for chemotherapy-induced hemorrhagic cystitis.

Chemotherapy-induced hemorrhagic cystitis (CHC) can be difficult to manage. Prior work suggests that IL-4 alleviates ifosfamide-induced hemorrhagic cystitis (IHC), but systemically administered IL-4 causes significant side effects. We hypothesized that the Schistosoma hematobium homolog of IL-4-inducing principle from Schistosoma mansoni eggs (H-IPSE), would reduce IHC and associated bladder pathology. IPSE binds IgE on basophils and mast cells, triggering IL-4 secretion by these cells. IPSE is also an “infiltrin,” translocating into the host nucleus to modulate gene transcription. Mice were administered IL-4, H-IPSE protein or its nuclear localization sequence (NLS) mutant, with or without neutralizing anti-IL-4 antibody, or 2-mercaptoethane sulfonate sodium (MESNA; a drug used to prevent IHC), followed by ifosfamide. Bladder tissue damage and hemoglobin content were measured. Spontaneous and evoked pain, urinary frequency, and bladdergene expression analysis were assessed. Pain behaviors were interpreted in a blinded fashion. One dose of H-IPSE was superior to MESNA and IL-4 in suppressing bladder hemorrhage in an IL-4-dependent fashion and comparable with MESNA in dampening ifosfamide-triggered pain behaviors in an NLS-dependent manner. H-IPSE also accelerated urothelial repair following IHC. Our work represents the first therapeutic exploitation of a uropathogen-derived host modulatory molecule in a clinically relevant bladder disease model and indicates that IPSE may be an alternative to MESNA for mitigating CHC.—Mbanefo, E. C., Le, L., Pennington, L. F., Odegaard, J. I., Jardetzky, T. S., Alouffi, A., Falcone, F. H., Hsieh, M. H. Therapeutic exploitation of IPSE, a urogenital parasite-derived host modulatory protein, for chemotherapy-induced hemorrhagic cystitis.

Sleeping beauty: awakening urothelium from its slumber.

The bladder urothelium is essentially quiescent but regenerates readily upon injury. The process of urothelial regeneration harkens back to the process of urothelial development whereby urothelial stem/progenitor cells must proliferate and terminally differentiate to establish all three urothelial layers. How the urothelium regulates the level of proliferation and the timing of differentiation to ensure the precise degree of regeneration is of significant interest in the field. Without a carefully-orchestrated process, urothelial regeneration may be inadequate, thereby exposing the host to toxins or pathogens. Alternatively, regeneration may be excessive, thereby setting the stage for tumor development. This review describes our current understanding of urothelial regeneration. The current controversies surrounding the identity and location of urothelial progenitor cells that mediate urothelial regeneration are discussed and evidence for each model is provided. We emphasize the factors that have been shown to be crucial for urothelial regeneration, including local growth factors that stimulate repair, and epithelial-mesenchymal cross talk, which ensures feedback regulation. Also highlighted is the emerging concept of epigenetic regulation of urothelial regeneration, which additionally fine tunes the process through transcriptional regulation of cell cycle genes and growth and differentiation factors. Finally, we emphasize how several of these pathways and/or programs are often dysregulated during malignant transformation, further corroborating their importance in directing normal urothelial regeneration. Together, evidence in the field suggests that any attempt to exploit regenerative programs for the purposes of enhanced urothelial repair or replacement must take into account this delicate balance.

Compensatory paracrine mechanisms that define the urothelial response to injury in partial bladder outlet obstruction

Diseases and conditions affecting the lower urinary tract are a leading cause of dysfunctional sexual health, incontinence, infection, and kidney failure. The growth, differentiation, and repair of the bladder's epithelial lining are regulated, in part, by fibroblast growth factor (FGF)-7 and -10 via a paracrine cascade originating in the mesenchyme (lamina propria) and targeting the receptor for FGF-7 and -10 within the transitional epithelium (urothelium). The FGF-7 gene is located at the 15q15-q21.1 locus on chromosome 15 and four exons generate a 3.852-kb mRNA. Five duplicated FGF-7 gene sequences that localized to chromosome 9 were predicted not to generate functional protein products, thus validating the use of FGF-7-null mice as an experimental model. Recombinant FGF-7 and -10 induced proliferation of human urothelial cells in vitro and transitional epithelium of wild-type and FGF-7-null mice in vivo. To determine the extent that induction of urothelial cell proliferation during the bladder response to injury is dependent on FGF-7, an animal model of partial bladder outlet obstruction was developed. Unbiased stereology was used to measure the percentage of proliferating urothelial cells between obstructed groups of wild-type and FGF-7-null mice. The stereological analysis indicated that a statistical significant difference did not exist between the two groups, suggesting that FGF-7 is not essential for urothelial cell proliferation in response to partial outlet obstruction. In contrast, a significant increase in FGF-10 expression was observed in the obstructed FGF-7-null group, indicating that the compensatory pathway that functions in this model results in urothelial repair.

Functions of epidermal growth factor-like growth factors during human urothelial reepithelialization in vitro and the role of erbB2.

Transitional epithelium of the urinary bladder can be damaged during, for example, catheterization, overstretching due to obstructed voiding, or partial resection. The subsequent repair process can be stimulated by specific proteins such as epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha). However, little is known about the role of EGF-like growth factors and their respective receptors in human urothelial repair. In this study, we examined the effects of EGF, TGFalpha, amphiregulin and heregulin-alpha (HRGalpha) on proliferation, wound closure, and the expression of their receptors c-erbB1-c-erbB4 in primary cultures of human urothelial cells in vitro. Under conditions representing intact urothelium, all EGF-like growth factors except HRGalpha induced proliferation. TGFalpha induced proliferation up to four times. Amphiregulin increased expression of c-erbB1. Treatment with either TGFalpha or amphiregulin resulted in higher c-erbB1 activation and c-erbB3 levels. None of the growth factors affected the constitutive expression of c-erbB2 and c-erbB4. In the repair model, both EGF and TGFalpha stimulated the wound closure most strongly. This was mainly achieved by increased cellular migration. Receptor expression was not affected by the addition of exogenous growth factor. The role of c-erbB2 in wound healing was further investigated with the use of antisense DNA. Wound closure could be delayed up to 50% by antisense c-erbB2 but not by mismatched or sense oligonucleotides. Excessive production (e.g. in bladder tumors) or application of EGF, TGFalpha or amphiregulin, but not HRGalpha may lead to either hyperplasia or a faster repair of damaged urothelium in vivo. These effects seem to be mediated not only via c-erbB1 but also via c-erbB2. Our results suggest that modified members of the EGF-EGFR family are potential targets for future therapies for bladder wound healing and malignancy.

The Use of an Extended Spiral Bladder Flap for Treatment of Upper Ureteral Loss

Purpose We demonstrated the effectiveness of an extended spiral bladder flap procedure for repair of upper ureteral injuries. Materials and Methods Two patients with extensive ureteral loss underwent repair with a modified bladder flap based posterolaterally on the affected side, spiraled anteriorly and inferolaterally toward the contralateral bladder base, and then anastomosed directly to the renal pelvis. Results With this technique renal function was preserved in both patients without obstruction on followup radiographs. Conclusions Using this extended bladder flap technique a well vascularized urothelial repair can be achieved for upper ureteral injury in select patients.