Soluble Guanylate Cyclase Activator, BAY 58-2667, Decreases Neurogenic Detrusor Overactivity and Reverses Bladder Fibrosis in Mice with Radiation Cystitis

Zabbarova I1, Ikeda Y1, Getchell S1, Kozlowski M1, Drake M2, Fry C2, Birder L1, Kanai A1

Research Type

Basic Science / Translational

Abstract Category

Overactive Bladder

Abstract 314
E-Poster 2
Scientific Open Discussion ePoster Session 18
Thursday 5th September 2019
13:15 - 13:20 (ePoster Station 3)
Exhibition Hall
Animal Study Pre-Clinical testing Pharmacology
1.University of Pittsburgh, 2.University of Bristol
Presenter
I

Irina Zabbarova

Links

Poster

Abstract

Hypothesis / aims of study
The role of nitric oxide (NO•) signalling in the urinary bladder is incompletely understood.  Early studies demonstrated NO•-mediated relaxation in the bladder neck and urethra and detrusor relaxation is generally thought to be mediated by adrenergic beta receptors.  However, there is robust expression of soluble guanylate cyclase (sGC) and NO•-induced cyclic guanosine monophosphate (cGMP) in the urothelium, vascular smooth muscle and interstitial cells of the bladder wall.  Furthermore, NO•-cGMP modulators dampen bladder afferent nerve firing [1].  NO• activates sGC by binding to its beta-subunit, inducing a conformational change converting GTP to cGMP.  A prerequisite for NO•-induced sGC activation is a reduced heme iron (Fe2+), as NO• cannot bind to sGC with oxidized heme (Fe3+).  Thus, oxidative stress conditions can attenuate NO•-mediated cGMP signalling.  sGC activators do not require NO•, which can be low or absent in conditions of nitrergic nerve damage, and act on sGC with oxidized heme (or even the absence of heme) in pathology where PDE-5 inhibitors (e.g., sildenafil/Viagra) are ineffective.  It has been hypothesised that cGMP signalling suppressing TGF-beta1 is involved in antifibrotic mechanisms [2] and the aim of this study was to investigate if sGC activators can reduce fibrosis and re-establish normal voiding function in mice with chronic radiation cystitis.
Study design, materials and methods
Adult female C57BL/6 mice were anesthetized with avertin, 300 mg/kg, had their urinary bladders externalized and selectively irradiated (10 Gy; 320 KV X-ray irradiator) without affecting other pelvic structures.  We have previously demonstrated that this procedure causes development of fibrosis four to six weeks later.  After seven weeks following irradiation, mice were gavaged for two weeks daily with BAY 58-2667 (10 mg/kg/day) or vehicle (0.5% methylcellulose and 10% DMSO).  Following treatment, bladder function was evaluated in vivo using decerebrate cystometrograms (CMGs) and in vitro using length-tension recordings from bladder strips.  Histological staining was used for bladder wall structure and collagen content observation.  Experiments were carried out on n ≥ 4 mice.  Unpaired student t-test determined differences between irradiated versus control groups or parameters with and without treatment.
Results
Mice with chronic radiation cystitis developed neurogenic detrusor overactivity (NDO) demonstrated as a significant decrease in intercontractile intervals, non-voiding contractions and decreased compliance in CMG recordings.  Daily gavage with BAY 58-2667 normalized the CMG profiles (Figure 1A).  Length-tension measurements from isolated bladder strips showed a significant decline in active force generation and increased passive tension/tissue stiffness in chronically irradiated mice compared to controls (Figure 1B).  Modified Verhoeff Van Gieson staining showed significant collagen deposition (Figure 1C) and increased collagen:tissue ratio as well as damaged urothelial layer suggestive of chronic or re-occurring inflammation.  Chronically irradiated mice treated with BAY 58-2667 had tension profiles and a bladder wall structure similar to non-irradiated controls (Figures 1B and 1C).
Interpretation of results
Our studies demonstrate that BAY 58-2667 can decrease NDO and reverse fibrosis in mice with chronic radiation cystitis.  BAY 58-2667 treatment decreased passive and increased active tension profiles in isolated bladder strips demonstrating improved bladder compliance and force generation.  This treatment also inhibited re-occurring inflammation permitting the urothelium to recover and re-establish barrier function.  These results support a role for NO•-mediated signalling in reversing irradiation-induced bladder fibrosis and improving bladder function.
Concluding message
A series of sGC activators have been developed as potential treatment for pulmonary hypertension and fibrosis, hypothetically acting through suppression of TGF-beta1, upregulation of matrix metalloproteinases and downregulation of tissue inhibitor of metalloproteinase.  There is also evidence that these compounds ameliorate cyclophosphamide-induced bladder overactivity which correlates with downregulation of bladder sGC expression [3] and may act directly on afferent nerves to dampen their excitability.  We have demonstrated that activation of sGC can reverse bladder fibrosis and rescue bladder function in chronic radiation cystitis.  Fibrosis is implicated in most benign bladder pathologies making a high clinical demand for agents ameliorating or reversing it.
Figure 1 The effect of sGC activator on bladder activity in chronic irradiation cystitis.
References
  1. Minagawa T, et al. BJU Int. 2012. 110(6B):E259-66. 22591258
  2. Ng HH, et al. Mol Cell Endocrinol. 2019. DOI: 10.1016/j.mce.2019.01.015. 30660699
  3. de Oliceira MG, et al. Am J Physiol Renal Physiol. 2016. 311(1):F85-93. 27122537
Disclosures
Funding Awards from NIH/NIDDK; R01 DK071085 (Kanai), R01 DK098361 (Kanai and Drake), P01 DK093424 (Kanai) and Department of Defense SC170171 (Kanai and Ikeda) Clinical Trial No Subjects Animal Species Mice Ethics Committee University of Pittsburgh Institutional Animal Care and Use Committee